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    Bio-Rad micro biospin p 30 gel columns bio rad
    Micro Biospin P 30 Gel Columns Bio Rad, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/micro+biospin+p+30+gel+columns+bio+rad/Micro+Bio-Spin+P-30+Gel+Columns/pm41232527-244-13-18
    Average 96 stars, based on 364 article reviews
    micro biospin p 30 gel columns bio rad - by Bioz Stars, 2026-09
    96/100 stars

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    Bicinchoninic Acid Protein Assay:

    Article Title: A role for human senataxin in contending with pausing and backtracking during transcript elongation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Puromycin Thermo Fisher Scientific Cat # A1113803 Geneticin (G418 Sulfate) (50 mg/mL) Thermo Fisher Scientific Cat # 10131035 Doxycycline Clontech Cat # 8634-1 4-thiouridine Glentham Life Sciences Cat # GN6085 4-thiouracil Sigma-Aldrich Cat # 440736 MTSEA biotin-XX linker Biotium Cat # BT90066 DynabeadsTM Protein G Invitrogen Cat # 10004D LipofectamineTM 3000 ThermoFisher Scientific Cat # 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat # 13778150 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat # D1916 dTAG-V1 Tocris Bioscience Cat # 6914 AMPureXP beads Beckman Coulter Cat # A63881 Biotinylated NTPs PerkinElmer Cat # NEL542001EA, Cat # NEL545001EA DynabeadsTM Protein A ThermoFisher Scientific Cat # 10002D RNase Inhibitor Murine NEB Cat # M0314L mRNA Decapping Enzyme NEB Cat # M0608S Adenosine 5’-Triphosphate (ATP) NEB Cat # P0756S T4 Polynucleotide Kinase (3’ phosphatase minus) NEB Cat # M0236S BaseMuncher Abcam Cat # ab270049 PhosSTOP Sigma-Aldrich Cat # 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 ANTI FLAG M2 magnetic beads Merckmillipore Cat # 8823 Mono QTM 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 NuPAGETM 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 Nitrocellulose Membrane, 0.45 μm Thermo Fisher Scientific Cat # 88018 PVDF Membrane Merckmillipore Cat # IPFL00010 (Continued on next page) Molecular Cell 85, 1–17.e1–e10, November 20, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat # 34580 Radiance plus Azure Biosystems Cat # AC2103 Poly-D-Lysine Thermo Fisher Scientific Cat # A3890401 5-Ethynyl Uridine (EU) Thermo Fisher Scientific Cat # 50-218-3520 THZ531 MedChemExpress Cat # HY-103618 Alexa Fluor 488 NHS Ester Thermo Fisher Scientific Cat # A20000 Formaldehyde Sigma-Aldrich Cat # F8775-25ML phi29 DNA Polymerase NEB Cat # M0269L Klenow Fragment (3’→5’ exo-) NEB Cat # M0212L T4 DNA Ligase NEB Cat # M0202M Critical commercial assays Malachite Green Phosphate Assay Kit Merckmillipore Cat# MAK307 Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat # 15778629 RNeasy minElute kit QIAGEN Cat # 74204 μMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Qubit RNA HS Assay Kit Thermo Fisher Cat # Q32852 Qubit RNA BR Assay Kit Thermo Fisher Cat # Q10210 KAPA RNA RiboErase HyperPrep kit Roche Cat # 8098131702 KAPA RNA HyperPrep Kit Roche Cat # 8098093702 NEBNext Ultra II Directional RNA Library Prep Kit for Illumina NEB Cat # E7760L NEBNext Multiplex Oligos for Illumina(Unique Dual Index UMI adapters RNA Set 1) NEB Cat # E7416S Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat # 12043795 Deposited data TT chem -seq This study GSE280010 DRBrelease_TT chem-seq This study GSE280011 PRO-seq This study GSE280012 R-ChIP-seq This study GSE280013 Hap1_TT chem -seq This study GSE297203 Hap1_DRBrelease_TT chem-seq This study GSE297204 INTS4-dTAG TT chem -seq Blears et al. 42 GSE253354 SETX ChIP-seq Cohen et al. 65 Array Express E-MTAB-6318 RNAPII ChIP-seq Kanagaraj et al. 24 GSE143574 CytoDRIP-seq Crossley et al. 82 GSE178841 Unprocessed and uncompressed imaging data This study https://doi.org/10.17632/yx6jk5zr44.1 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Hek293 Flp-In T-Rex ThermoFisher Scientific Cat # R78007 Hek293 Flp-In T-REx SETX-Dtag This study cZH9.1 Hek293 Flp-In T-Rex SETX-dTAG: SETX-mScarlet (flpin) This study cZH18 Hek293 Flp-In T-Rex SETX-dTAG: SETX-flag (flpin) This study cZH20 Hek293 Flp-In T-Rex SETX-dTAG: RNaseH1Mut(D210N)-V5 (flpin) This study cZH21 (Continued on next page) e3 Molecular Cell 85, 1–17.e1–e10, November 20, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hap1 SETX-Dtag This study cZH36.5 Experimental models: Organisms/strains Rpb3-flag::NAT, prb1Δ Kind gift from John Diffley N/A Oligonucleotides See Table S1 for Oligonucleotides This study N/A Recombinant DNA Plib Weissmann et al. 104 RRID: Addgene_80610 pLIB flag-SETXHD(1690-2677) This study pZH219 pLIB flag-SETXMut(1690-2677,D2181E2182-AA) This study pZH220 pET21b flag-SEN1HD(1095-1904) This study pZH235 pET21b flag-IGHMBP2HD(1-652) This study pZH233 pCRIS-HDR-SETX-dTAG-Puro This study pZH169 sgRNA against SETX in SpCas9-2A-EGFP for dTAG knock in This study pZH172 pcDNA5 FRT TO SETX-mScarlet-flag This study pZH199 pcDNA5 FRT TO SETX-flag This study pZH216 pcDNA5 FRT TO RNaseH1Mut(D210N)-V5 This study pZH217 SpCas9-2A-EGFP and single guide RNA Ran et al. 105 PX458 pOG44 ThermoFisher Scientific Cat # V600520 pCRIS-PITChv2_BRD3C-dTAG-Puro Nabet et al. 57 pZH154, RRID: Addgene_91796 pmScarlet_C1 Bindels et al. 106 pZH175, RRID: Addgene_85042 pcDNA5 FRT TO ThermoFisher Scientific V652020 pcDNA5 FRT TO (Geneticin) kind gift from Jonathon Pines pZH189, RRID: Addgene_41000 ppyCAG_RNaseH1_D210N Chen et al. 85 pZH206, RRID: Addgene_111904 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Fiji Schindelin et al. 107 https://imagej.net/Fiji UCSF Chimera X Goddard et al. 108 https://www.cgl.ucsf.edu/chimerax/ RStudio RStudio Team (2021).

    RNA HS Assay:

    Article Title: A role for human senataxin in contending with pausing and backtracking during transcript elongation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Puromycin Thermo Fisher Scientific Cat # A1113803 Geneticin (G418 Sulfate) (50 mg/mL) Thermo Fisher Scientific Cat # 10131035 Doxycycline Clontech Cat # 8634-1 4-thiouridine Glentham Life Sciences Cat # GN6085 4-thiouracil Sigma-Aldrich Cat # 440736 MTSEA biotin-XX linker Biotium Cat # BT90066 DynabeadsTM Protein G Invitrogen Cat # 10004D LipofectamineTM 3000 ThermoFisher Scientific Cat # 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat # 13778150 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat # D1916 dTAG-V1 Tocris Bioscience Cat # 6914 AMPureXP beads Beckman Coulter Cat # A63881 Biotinylated NTPs PerkinElmer Cat # NEL542001EA, Cat # NEL545001EA DynabeadsTM Protein A ThermoFisher Scientific Cat # 10002D RNase Inhibitor Murine NEB Cat # M0314L mRNA Decapping Enzyme NEB Cat # M0608S Adenosine 5’-Triphosphate (ATP) NEB Cat # P0756S T4 Polynucleotide Kinase (3’ phosphatase minus) NEB Cat # M0236S BaseMuncher Abcam Cat # ab270049 PhosSTOP Sigma-Aldrich Cat # 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 ANTI FLAG M2 magnetic beads Merckmillipore Cat # 8823 Mono QTM 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 NuPAGETM 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 Nitrocellulose Membrane, 0.45 μm Thermo Fisher Scientific Cat # 88018 PVDF Membrane Merckmillipore Cat # IPFL00010 (Continued on next page) Molecular Cell 85, 1–17.e1–e10, November 20, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat # 34580 Radiance plus Azure Biosystems Cat # AC2103 Poly-D-Lysine Thermo Fisher Scientific Cat # A3890401 5-Ethynyl Uridine (EU) Thermo Fisher Scientific Cat # 50-218-3520 THZ531 MedChemExpress Cat # HY-103618 Alexa Fluor 488 NHS Ester Thermo Fisher Scientific Cat # A20000 Formaldehyde Sigma-Aldrich Cat # F8775-25ML phi29 DNA Polymerase NEB Cat # M0269L Klenow Fragment (3’→5’ exo-) NEB Cat # M0212L T4 DNA Ligase NEB Cat # M0202M Critical commercial assays Malachite Green Phosphate Assay Kit Merckmillipore Cat# MAK307 Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat # 15778629 RNeasy minElute kit QIAGEN Cat # 74204 μMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Qubit RNA HS Assay Kit Thermo Fisher Cat # Q32852 Qubit RNA BR Assay Kit Thermo Fisher Cat # Q10210 KAPA RNA RiboErase HyperPrep kit Roche Cat # 8098131702 KAPA RNA HyperPrep Kit Roche Cat # 8098093702 NEBNext Ultra II Directional RNA Library Prep Kit for Illumina NEB Cat # E7760L NEBNext Multiplex Oligos for Illumina(Unique Dual Index UMI adapters RNA Set 1) NEB Cat # E7416S Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat # 12043795 Deposited data TT chem -seq This study GSE280010 DRBrelease_TT chem-seq This study GSE280011 PRO-seq This study GSE280012 R-ChIP-seq This study GSE280013 Hap1_TT chem -seq This study GSE297203 Hap1_DRBrelease_TT chem-seq This study GSE297204 INTS4-dTAG TT chem -seq Blears et al. 42 GSE253354 SETX ChIP-seq Cohen et al. 65 Array Express E-MTAB-6318 RNAPII ChIP-seq Kanagaraj et al. 24 GSE143574 CytoDRIP-seq Crossley et al. 82 GSE178841 Unprocessed and uncompressed imaging data This study https://doi.org/10.17632/yx6jk5zr44.1 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Hek293 Flp-In T-Rex ThermoFisher Scientific Cat # R78007 Hek293 Flp-In T-REx SETX-Dtag This study cZH9.1 Hek293 Flp-In T-Rex SETX-dTAG: SETX-mScarlet (flpin) This study cZH18 Hek293 Flp-In T-Rex SETX-dTAG: SETX-flag (flpin) This study cZH20 Hek293 Flp-In T-Rex SETX-dTAG: RNaseH1Mut(D210N)-V5 (flpin) This study cZH21 (Continued on next page) e3 Molecular Cell 85, 1–17.e1–e10, November 20, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hap1 SETX-Dtag This study cZH36.5 Experimental models: Organisms/strains Rpb3-flag::NAT, prb1Δ Kind gift from John Diffley N/A Oligonucleotides See Table S1 for Oligonucleotides This study N/A Recombinant DNA Plib Weissmann et al. 104 RRID: Addgene_80610 pLIB flag-SETXHD(1690-2677) This study pZH219 pLIB flag-SETXMut(1690-2677,D2181E2182-AA) This study pZH220 pET21b flag-SEN1HD(1095-1904) This study pZH235 pET21b flag-IGHMBP2HD(1-652) This study pZH233 pCRIS-HDR-SETX-dTAG-Puro This study pZH169 sgRNA against SETX in SpCas9-2A-EGFP for dTAG knock in This study pZH172 pcDNA5 FRT TO SETX-mScarlet-flag This study pZH199 pcDNA5 FRT TO SETX-flag This study pZH216 pcDNA5 FRT TO RNaseH1Mut(D210N)-V5 This study pZH217 SpCas9-2A-EGFP and single guide RNA Ran et al. 105 PX458 pOG44 ThermoFisher Scientific Cat # V600520 pCRIS-PITChv2_BRD3C-dTAG-Puro Nabet et al. 57 pZH154, RRID: Addgene_91796 pmScarlet_C1 Bindels et al. 106 pZH175, RRID: Addgene_85042 pcDNA5 FRT TO ThermoFisher Scientific V652020 pcDNA5 FRT TO (Geneticin) kind gift from Jonathon Pines pZH189, RRID: Addgene_41000 ppyCAG_RNaseH1_D210N Chen et al. 85 pZH206, RRID: Addgene_111904 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Fiji Schindelin et al. 107 https://imagej.net/Fiji UCSF Chimera X Goddard et al. 108 https://www.cgl.ucsf.edu/chimerax/ RStudio RStudio Team (2021).

    Multiplex Assay:

    Article Title: A role for human senataxin in contending with pausing and backtracking during transcript elongation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Puromycin Thermo Fisher Scientific Cat # A1113803 Geneticin (G418 Sulfate) (50 mg/mL) Thermo Fisher Scientific Cat # 10131035 Doxycycline Clontech Cat # 8634-1 4-thiouridine Glentham Life Sciences Cat # GN6085 4-thiouracil Sigma-Aldrich Cat # 440736 MTSEA biotin-XX linker Biotium Cat # BT90066 DynabeadsTM Protein G Invitrogen Cat # 10004D LipofectamineTM 3000 ThermoFisher Scientific Cat # 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat # 13778150 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat # D1916 dTAG-V1 Tocris Bioscience Cat # 6914 AMPureXP beads Beckman Coulter Cat # A63881 Biotinylated NTPs PerkinElmer Cat # NEL542001EA, Cat # NEL545001EA DynabeadsTM Protein A ThermoFisher Scientific Cat # 10002D RNase Inhibitor Murine NEB Cat # M0314L mRNA Decapping Enzyme NEB Cat # M0608S Adenosine 5’-Triphosphate (ATP) NEB Cat # P0756S T4 Polynucleotide Kinase (3’ phosphatase minus) NEB Cat # M0236S BaseMuncher Abcam Cat # ab270049 PhosSTOP Sigma-Aldrich Cat # 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 ANTI FLAG M2 magnetic beads Merckmillipore Cat # 8823 Mono QTM 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 NuPAGETM 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 Nitrocellulose Membrane, 0.45 μm Thermo Fisher Scientific Cat # 88018 PVDF Membrane Merckmillipore Cat # IPFL00010 (Continued on next page) Molecular Cell 85, 1–17.e1–e10, November 20, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat # 34580 Radiance plus Azure Biosystems Cat # AC2103 Poly-D-Lysine Thermo Fisher Scientific Cat # A3890401 5-Ethynyl Uridine (EU) Thermo Fisher Scientific Cat # 50-218-3520 THZ531 MedChemExpress Cat # HY-103618 Alexa Fluor 488 NHS Ester Thermo Fisher Scientific Cat # A20000 Formaldehyde Sigma-Aldrich Cat # F8775-25ML phi29 DNA Polymerase NEB Cat # M0269L Klenow Fragment (3’→5’ exo-) NEB Cat # M0212L T4 DNA Ligase NEB Cat # M0202M Critical commercial assays Malachite Green Phosphate Assay Kit Merckmillipore Cat# MAK307 Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat # 15778629 RNeasy minElute kit QIAGEN Cat # 74204 μMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Qubit RNA HS Assay Kit Thermo Fisher Cat # Q32852 Qubit RNA BR Assay Kit Thermo Fisher Cat # Q10210 KAPA RNA RiboErase HyperPrep kit Roche Cat # 8098131702 KAPA RNA HyperPrep Kit Roche Cat # 8098093702 NEBNext Ultra II Directional RNA Library Prep Kit for Illumina NEB Cat # E7760L NEBNext Multiplex Oligos for Illumina(Unique Dual Index UMI adapters RNA Set 1) NEB Cat # E7416S Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat # 12043795 Deposited data TT chem -seq This study GSE280010 DRBrelease_TT chem-seq This study GSE280011 PRO-seq This study GSE280012 R-ChIP-seq This study GSE280013 Hap1_TT chem -seq This study GSE297203 Hap1_DRBrelease_TT chem-seq This study GSE297204 INTS4-dTAG TT chem -seq Blears et al. 42 GSE253354 SETX ChIP-seq Cohen et al. 65 Array Express E-MTAB-6318 RNAPII ChIP-seq Kanagaraj et al. 24 GSE143574 CytoDRIP-seq Crossley et al. 82 GSE178841 Unprocessed and uncompressed imaging data This study https://doi.org/10.17632/yx6jk5zr44.1 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Hek293 Flp-In T-Rex ThermoFisher Scientific Cat # R78007 Hek293 Flp-In T-REx SETX-Dtag This study cZH9.1 Hek293 Flp-In T-Rex SETX-dTAG: SETX-mScarlet (flpin) This study cZH18 Hek293 Flp-In T-Rex SETX-dTAG: SETX-flag (flpin) This study cZH20 Hek293 Flp-In T-Rex SETX-dTAG: RNaseH1Mut(D210N)-V5 (flpin) This study cZH21 (Continued on next page) e3 Molecular Cell 85, 1–17.e1–e10, November 20, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hap1 SETX-Dtag This study cZH36.5 Experimental models: Organisms/strains Rpb3-flag::NAT, prb1Δ Kind gift from John Diffley N/A Oligonucleotides See Table S1 for Oligonucleotides This study N/A Recombinant DNA Plib Weissmann et al. 104 RRID: Addgene_80610 pLIB flag-SETXHD(1690-2677) This study pZH219 pLIB flag-SETXMut(1690-2677,D2181E2182-AA) This study pZH220 pET21b flag-SEN1HD(1095-1904) This study pZH235 pET21b flag-IGHMBP2HD(1-652) This study pZH233 pCRIS-HDR-SETX-dTAG-Puro This study pZH169 sgRNA against SETX in SpCas9-2A-EGFP for dTAG knock in This study pZH172 pcDNA5 FRT TO SETX-mScarlet-flag This study pZH199 pcDNA5 FRT TO SETX-flag This study pZH216 pcDNA5 FRT TO RNaseH1Mut(D210N)-V5 This study pZH217 SpCas9-2A-EGFP and single guide RNA Ran et al. 105 PX458 pOG44 ThermoFisher Scientific Cat # V600520 pCRIS-PITChv2_BRD3C-dTAG-Puro Nabet et al. 57 pZH154, RRID: Addgene_91796 pmScarlet_C1 Bindels et al. 106 pZH175, RRID: Addgene_85042 pcDNA5 FRT TO ThermoFisher Scientific V652020 pcDNA5 FRT TO (Geneticin) kind gift from Jonathon Pines pZH189, RRID: Addgene_41000 ppyCAG_RNaseH1_D210N Chen et al. 85 pZH206, RRID: Addgene_111904 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Fiji Schindelin et al. 107 https://imagej.net/Fiji UCSF Chimera X Goddard et al. 108 https://www.cgl.ucsf.edu/chimerax/ RStudio RStudio Team (2021).

    Flow Cytometry:

    Article Title: A role for human senataxin in contending with pausing and backtracking during transcript elongation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Puromycin Thermo Fisher Scientific Cat # A1113803 Geneticin (G418 Sulfate) (50 mg/mL) Thermo Fisher Scientific Cat # 10131035 Doxycycline Clontech Cat # 8634-1 4-thiouridine Glentham Life Sciences Cat # GN6085 4-thiouracil Sigma-Aldrich Cat # 440736 MTSEA biotin-XX linker Biotium Cat # BT90066 DynabeadsTM Protein G Invitrogen Cat # 10004D LipofectamineTM 3000 ThermoFisher Scientific Cat # 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat # 13778150 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat # D1916 dTAG-V1 Tocris Bioscience Cat # 6914 AMPureXP beads Beckman Coulter Cat # A63881 Biotinylated NTPs PerkinElmer Cat # NEL542001EA, Cat # NEL545001EA DynabeadsTM Protein A ThermoFisher Scientific Cat # 10002D RNase Inhibitor Murine NEB Cat # M0314L mRNA Decapping Enzyme NEB Cat # M0608S Adenosine 5’-Triphosphate (ATP) NEB Cat # P0756S T4 Polynucleotide Kinase (3’ phosphatase minus) NEB Cat # M0236S BaseMuncher Abcam Cat # ab270049 PhosSTOP Sigma-Aldrich Cat # 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 ANTI FLAG M2 magnetic beads Merckmillipore Cat # 8823 Mono QTM 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 NuPAGETM 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 Nitrocellulose Membrane, 0.45 μm Thermo Fisher Scientific Cat # 88018 PVDF Membrane Merckmillipore Cat # IPFL00010 (Continued on next page) Molecular Cell 85, 1–17.e1–e10, November 20, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat # 34580 Radiance plus Azure Biosystems Cat # AC2103 Poly-D-Lysine Thermo Fisher Scientific Cat # A3890401 5-Ethynyl Uridine (EU) Thermo Fisher Scientific Cat # 50-218-3520 THZ531 MedChemExpress Cat # HY-103618 Alexa Fluor 488 NHS Ester Thermo Fisher Scientific Cat # A20000 Formaldehyde Sigma-Aldrich Cat # F8775-25ML phi29 DNA Polymerase NEB Cat # M0269L Klenow Fragment (3’→5’ exo-) NEB Cat # M0212L T4 DNA Ligase NEB Cat # M0202M Critical commercial assays Malachite Green Phosphate Assay Kit Merckmillipore Cat# MAK307 Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat # 15778629 RNeasy minElute kit QIAGEN Cat # 74204 μMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Qubit RNA HS Assay Kit Thermo Fisher Cat # Q32852 Qubit RNA BR Assay Kit Thermo Fisher Cat # Q10210 KAPA RNA RiboErase HyperPrep kit Roche Cat # 8098131702 KAPA RNA HyperPrep Kit Roche Cat # 8098093702 NEBNext Ultra II Directional RNA Library Prep Kit for Illumina NEB Cat # E7760L NEBNext Multiplex Oligos for Illumina(Unique Dual Index UMI adapters RNA Set 1) NEB Cat # E7416S Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat # 12043795 Deposited data TT chem -seq This study GSE280010 DRBrelease_TT chem-seq This study GSE280011 PRO-seq This study GSE280012 R-ChIP-seq This study GSE280013 Hap1_TT chem -seq This study GSE297203 Hap1_DRBrelease_TT chem-seq This study GSE297204 INTS4-dTAG TT chem -seq Blears et al. 42 GSE253354 SETX ChIP-seq Cohen et al. 65 Array Express E-MTAB-6318 RNAPII ChIP-seq Kanagaraj et al. 24 GSE143574 CytoDRIP-seq Crossley et al. 82 GSE178841 Unprocessed and uncompressed imaging data This study https://doi.org/10.17632/yx6jk5zr44.1 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Hek293 Flp-In T-Rex ThermoFisher Scientific Cat # R78007 Hek293 Flp-In T-REx SETX-Dtag This study cZH9.1 Hek293 Flp-In T-Rex SETX-dTAG: SETX-mScarlet (flpin) This study cZH18 Hek293 Flp-In T-Rex SETX-dTAG: SETX-flag (flpin) This study cZH20 Hek293 Flp-In T-Rex SETX-dTAG: RNaseH1Mut(D210N)-V5 (flpin) This study cZH21 (Continued on next page) e3 Molecular Cell 85, 1–17.e1–e10, November 20, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hap1 SETX-Dtag This study cZH36.5 Experimental models: Organisms/strains Rpb3-flag::NAT, prb1Δ Kind gift from John Diffley N/A Oligonucleotides See Table S1 for Oligonucleotides This study N/A Recombinant DNA Plib Weissmann et al. 104 RRID: Addgene_80610 pLIB flag-SETXHD(1690-2677) This study pZH219 pLIB flag-SETXMut(1690-2677,D2181E2182-AA) This study pZH220 pET21b flag-SEN1HD(1095-1904) This study pZH235 pET21b flag-IGHMBP2HD(1-652) This study pZH233 pCRIS-HDR-SETX-dTAG-Puro This study pZH169 sgRNA against SETX in SpCas9-2A-EGFP for dTAG knock in This study pZH172 pcDNA5 FRT TO SETX-mScarlet-flag This study pZH199 pcDNA5 FRT TO SETX-flag This study pZH216 pcDNA5 FRT TO RNaseH1Mut(D210N)-V5 This study pZH217 SpCas9-2A-EGFP and single guide RNA Ran et al. 105 PX458 pOG44 ThermoFisher Scientific Cat # V600520 pCRIS-PITChv2_BRD3C-dTAG-Puro Nabet et al. 57 pZH154, RRID: Addgene_91796 pmScarlet_C1 Bindels et al. 106 pZH175, RRID: Addgene_85042 pcDNA5 FRT TO ThermoFisher Scientific V652020 pcDNA5 FRT TO (Geneticin) kind gift from Jonathon Pines pZH189, RRID: Addgene_41000 ppyCAG_RNaseH1_D210N Chen et al. 85 pZH206, RRID: Addgene_111904 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Fiji Schindelin et al. 107 https://imagej.net/Fiji UCSF Chimera X Goddard et al. 108 https://www.cgl.ucsf.edu/chimerax/ RStudio RStudio Team (2021).

    Chromatin Immunoprecipitation:

    Article Title: A role for human senataxin in contending with pausing and backtracking during transcript elongation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Puromycin Thermo Fisher Scientific Cat # A1113803 Geneticin (G418 Sulfate) (50 mg/mL) Thermo Fisher Scientific Cat # 10131035 Doxycycline Clontech Cat # 8634-1 4-thiouridine Glentham Life Sciences Cat # GN6085 4-thiouracil Sigma-Aldrich Cat # 440736 MTSEA biotin-XX linker Biotium Cat # BT90066 DynabeadsTM Protein G Invitrogen Cat # 10004D LipofectamineTM 3000 ThermoFisher Scientific Cat # 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat # 13778150 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat # D1916 dTAG-V1 Tocris Bioscience Cat # 6914 AMPureXP beads Beckman Coulter Cat # A63881 Biotinylated NTPs PerkinElmer Cat # NEL542001EA, Cat # NEL545001EA DynabeadsTM Protein A ThermoFisher Scientific Cat # 10002D RNase Inhibitor Murine NEB Cat # M0314L mRNA Decapping Enzyme NEB Cat # M0608S Adenosine 5’-Triphosphate (ATP) NEB Cat # P0756S T4 Polynucleotide Kinase (3’ phosphatase minus) NEB Cat # M0236S BaseMuncher Abcam Cat # ab270049 PhosSTOP Sigma-Aldrich Cat # 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 ANTI FLAG M2 magnetic beads Merckmillipore Cat # 8823 Mono QTM 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 NuPAGETM 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 Nitrocellulose Membrane, 0.45 μm Thermo Fisher Scientific Cat # 88018 PVDF Membrane Merckmillipore Cat # IPFL00010 (Continued on next page) Molecular Cell 85, 1–17.e1–e10, November 20, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat # 34580 Radiance plus Azure Biosystems Cat # AC2103 Poly-D-Lysine Thermo Fisher Scientific Cat # A3890401 5-Ethynyl Uridine (EU) Thermo Fisher Scientific Cat # 50-218-3520 THZ531 MedChemExpress Cat # HY-103618 Alexa Fluor 488 NHS Ester Thermo Fisher Scientific Cat # A20000 Formaldehyde Sigma-Aldrich Cat # F8775-25ML phi29 DNA Polymerase NEB Cat # M0269L Klenow Fragment (3’→5’ exo-) NEB Cat # M0212L T4 DNA Ligase NEB Cat # M0202M Critical commercial assays Malachite Green Phosphate Assay Kit Merckmillipore Cat# MAK307 Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat # 15778629 RNeasy minElute kit QIAGEN Cat # 74204 μMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Qubit RNA HS Assay Kit Thermo Fisher Cat # Q32852 Qubit RNA BR Assay Kit Thermo Fisher Cat # Q10210 KAPA RNA RiboErase HyperPrep kit Roche Cat # 8098131702 KAPA RNA HyperPrep Kit Roche Cat # 8098093702 NEBNext Ultra II Directional RNA Library Prep Kit for Illumina NEB Cat # E7760L NEBNext Multiplex Oligos for Illumina(Unique Dual Index UMI adapters RNA Set 1) NEB Cat # E7416S Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat # 12043795 Deposited data TT chem -seq This study GSE280010 DRBrelease_TT chem-seq This study GSE280011 PRO-seq This study GSE280012 R-ChIP-seq This study GSE280013 Hap1_TT chem -seq This study GSE297203 Hap1_DRBrelease_TT chem-seq This study GSE297204 INTS4-dTAG TT chem -seq Blears et al. 42 GSE253354 SETX ChIP-seq Cohen et al. 65 Array Express E-MTAB-6318 RNAPII ChIP-seq Kanagaraj et al. 24 GSE143574 CytoDRIP-seq Crossley et al. 82 GSE178841 Unprocessed and uncompressed imaging data This study https://doi.org/10.17632/yx6jk5zr44.1 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Hek293 Flp-In T-Rex ThermoFisher Scientific Cat # R78007 Hek293 Flp-In T-REx SETX-Dtag This study cZH9.1 Hek293 Flp-In T-Rex SETX-dTAG: SETX-mScarlet (flpin) This study cZH18 Hek293 Flp-In T-Rex SETX-dTAG: SETX-flag (flpin) This study cZH20 Hek293 Flp-In T-Rex SETX-dTAG: RNaseH1Mut(D210N)-V5 (flpin) This study cZH21 (Continued on next page) e3 Molecular Cell 85, 1–17.e1–e10, November 20, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hap1 SETX-Dtag This study cZH36.5 Experimental models: Organisms/strains Rpb3-flag::NAT, prb1Δ Kind gift from John Diffley N/A Oligonucleotides See Table S1 for Oligonucleotides This study N/A Recombinant DNA Plib Weissmann et al. 104 RRID: Addgene_80610 pLIB flag-SETXHD(1690-2677) This study pZH219 pLIB flag-SETXMut(1690-2677,D2181E2182-AA) This study pZH220 pET21b flag-SEN1HD(1095-1904) This study pZH235 pET21b flag-IGHMBP2HD(1-652) This study pZH233 pCRIS-HDR-SETX-dTAG-Puro This study pZH169 sgRNA against SETX in SpCas9-2A-EGFP for dTAG knock in This study pZH172 pcDNA5 FRT TO SETX-mScarlet-flag This study pZH199 pcDNA5 FRT TO SETX-flag This study pZH216 pcDNA5 FRT TO RNaseH1Mut(D210N)-V5 This study pZH217 SpCas9-2A-EGFP and single guide RNA Ran et al. 105 PX458 pOG44 ThermoFisher Scientific Cat # V600520 pCRIS-PITChv2_BRD3C-dTAG-Puro Nabet et al. 57 pZH154, RRID: Addgene_91796 pmScarlet_C1 Bindels et al. 106 pZH175, RRID: Addgene_85042 pcDNA5 FRT TO ThermoFisher Scientific V652020 pcDNA5 FRT TO (Geneticin) kind gift from Jonathon Pines pZH189, RRID: Addgene_41000 ppyCAG_RNaseH1_D210N Chen et al. 85 pZH206, RRID: Addgene_111904 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Fiji Schindelin et al. 107 https://imagej.net/Fiji UCSF Chimera X Goddard et al. 108 https://www.cgl.ucsf.edu/chimerax/ RStudio RStudio Team (2021).

    Imaging:

    Article Title: A role for human senataxin in contending with pausing and backtracking during transcript elongation.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Puromycin Thermo Fisher Scientific Cat # A1113803 Geneticin (G418 Sulfate) (50 mg/mL) Thermo Fisher Scientific Cat # 10131035 Doxycycline Clontech Cat # 8634-1 4-thiouridine Glentham Life Sciences Cat # GN6085 4-thiouracil Sigma-Aldrich Cat # 440736 MTSEA biotin-XX linker Biotium Cat # BT90066 DynabeadsTM Protein G Invitrogen Cat # 10004D LipofectamineTM 3000 ThermoFisher Scientific Cat # 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat # 13778150 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat # D1916 dTAG-V1 Tocris Bioscience Cat # 6914 AMPureXP beads Beckman Coulter Cat # A63881 Biotinylated NTPs PerkinElmer Cat # NEL542001EA, Cat # NEL545001EA DynabeadsTM Protein A ThermoFisher Scientific Cat # 10002D RNase Inhibitor Murine NEB Cat # M0314L mRNA Decapping Enzyme NEB Cat # M0608S Adenosine 5’-Triphosphate (ATP) NEB Cat # P0756S T4 Polynucleotide Kinase (3’ phosphatase minus) NEB Cat # M0236S BaseMuncher Abcam Cat # ab270049 PhosSTOP Sigma-Aldrich Cat # 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 ANTI FLAG M2 magnetic beads Merckmillipore Cat # 8823 Mono QTM 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 NuPAGETM 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 Nitrocellulose Membrane, 0.45 μm Thermo Fisher Scientific Cat # 88018 PVDF Membrane Merckmillipore Cat # IPFL00010 (Continued on next page) Molecular Cell 85, 1–17.e1–e10, November 20, 2025 e2 .. REAGENT or RESOURCE SOURCE IDENTIFIER Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat # 34580 Radiance plus Azure Biosystems Cat # AC2103 Poly-D-Lysine Thermo Fisher Scientific Cat # A3890401 5-Ethynyl Uridine (EU) Thermo Fisher Scientific Cat # 50-218-3520 THZ531 MedChemExpress Cat # HY-103618 Alexa Fluor 488 NHS Ester Thermo Fisher Scientific Cat # A20000 Formaldehyde Sigma-Aldrich Cat # F8775-25ML phi29 DNA Polymerase NEB Cat # M0269L Klenow Fragment (3’→5’ exo-) NEB Cat # M0212L T4 DNA Ligase NEB Cat # M0202M Critical commercial assays Malachite Green Phosphate Assay Kit Merckmillipore Cat# MAK307 Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat # 15778629 RNeasy minElute kit QIAGEN Cat # 74204 μMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Qubit RNA HS Assay Kit Thermo Fisher Cat # Q32852 Qubit RNA BR Assay Kit Thermo Fisher Cat # Q10210 KAPA RNA RiboErase HyperPrep kit Roche Cat # 8098131702 KAPA RNA HyperPrep Kit Roche Cat # 8098093702 NEBNext Ultra II Directional RNA Library Prep Kit for Illumina NEB Cat # E7760L NEBNext Multiplex Oligos for Illumina(Unique Dual Index UMI adapters RNA Set 1) NEB Cat # E7416S Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat # 12043795 Deposited data TT chem -seq This study GSE280010 DRBrelease_TT chem-seq This study GSE280011 PRO-seq This study GSE280012 R-ChIP-seq This study GSE280013 Hap1_TT chem -seq This study GSE297203 Hap1_DRBrelease_TT chem-seq This study GSE297204 INTS4-dTAG TT chem -seq Blears et al. 42 GSE253354 SETX ChIP-seq Cohen et al. 65 Array Express E-MTAB-6318 RNAPII ChIP-seq Kanagaraj et al. 24 GSE143574 CytoDRIP-seq Crossley et al. 82 GSE178841 Unprocessed and uncompressed imaging data This study https://doi.org/10.17632/yx6jk5zr44.1 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Hek293 Flp-In T-Rex ThermoFisher Scientific Cat # R78007 Hek293 Flp-In T-REx SETX-Dtag This study cZH9.1 Hek293 Flp-In T-Rex SETX-dTAG: SETX-mScarlet (flpin) This study cZH18 Hek293 Flp-In T-Rex SETX-dTAG: SETX-flag (flpin) This study cZH20 Hek293 Flp-In T-Rex SETX-dTAG: RNaseH1Mut(D210N)-V5 (flpin) This study cZH21 (Continued on next page) e3 Molecular Cell 85, 1–17.e1–e10, November 20, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Hap1 SETX-Dtag This study cZH36.5 Experimental models: Organisms/strains Rpb3-flag::NAT, prb1Δ Kind gift from John Diffley N/A Oligonucleotides See Table S1 for Oligonucleotides This study N/A Recombinant DNA Plib Weissmann et al. 104 RRID: Addgene_80610 pLIB flag-SETXHD(1690-2677) This study pZH219 pLIB flag-SETXMut(1690-2677,D2181E2182-AA) This study pZH220 pET21b flag-SEN1HD(1095-1904) This study pZH235 pET21b flag-IGHMBP2HD(1-652) This study pZH233 pCRIS-HDR-SETX-dTAG-Puro This study pZH169 sgRNA against SETX in SpCas9-2A-EGFP for dTAG knock in This study pZH172 pcDNA5 FRT TO SETX-mScarlet-flag This study pZH199 pcDNA5 FRT TO SETX-flag This study pZH216 pcDNA5 FRT TO RNaseH1Mut(D210N)-V5 This study pZH217 SpCas9-2A-EGFP and single guide RNA Ran et al. 105 PX458 pOG44 ThermoFisher Scientific Cat # V600520 pCRIS-PITChv2_BRD3C-dTAG-Puro Nabet et al. 57 pZH154, RRID: Addgene_91796 pmScarlet_C1 Bindels et al. 106 pZH175, RRID: Addgene_85042 pcDNA5 FRT TO ThermoFisher Scientific V652020 pcDNA5 FRT TO (Geneticin) kind gift from Jonathon Pines pZH189, RRID: Addgene_41000 ppyCAG_RNaseH1_D210N Chen et al. 85 pZH206, RRID: Addgene_111904 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Fiji Schindelin et al. 107 https://imagej.net/Fiji UCSF Chimera X Goddard et al. 108 https://www.cgl.ucsf.edu/chimerax/ RStudio RStudio Team (2021).

    Blocking Assay:

    Article Title: DNA-directed termination of RNA polymerase II transcription.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-RNAPII subunit Rpb1 (8WG16) Cell services, The Francis Crick Institute N/A Donkey Anti-mouse, HRP conjugate Jackson ImmunoResearch Cat # 715-035-151; RRID: AB_2340771 Donkey Anti-mouse, Alexa Fluor 680 conjugate Invitrogen Cat # A10038; RRID: AB_2534014 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat # C2988J BL21-CodonPlus (DE3)-RIPL Thermo Fisher Scientific Cat # NC9122855 DH10Bac Competent Cells Thermo Fisher Scientific Cat # 10361012 Chemicals, peptides, and recombinant proteins complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 Mono Q 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L PolyA Polymerase from E.coli NEB Cat # M0276L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L PrimeSTAR HS with GC buffer Takara Cat # R044B HisTrap FF Crude Cytiva Cat # 11000458 HiTrap Heparin HP Cytiva Cat # 17040601 Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 RNase T1 Thermo Fisher Scientific Cat # EN0541 Invitrogen 0.1 to 2kb RNA Ladder Thermo Fisher Scientific Cat # 11518766 RNA marker templates Thermo Fisher Scientific Cat # AM7782 NuPAGE 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 PVDF Membrane Merckmillipore Cat # IPFL00010 4-thiouracil Sigma-Aldrich Cat # 440736 Monoclonal ANTI-FLAG M2 antibody Sigma-Aldrich Cat # F3165-1MG (Continued on next page) e1 Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 .. REAGENT or RESOURCE SOURCE IDENTIFIER Rapamycin Sigma-Aldrich Cat # R0395 MTSEA biotin-XX linker Biotium Cat # BT90066 Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 Intercept (PBS) Blocking Buffer LI-COR Cat # 927-70001 Fluorescein-12-UTP Jena bioscience Cat # NU-821-FAMX Critical commercial assays Pierce 660nm Protein Assay Reagent Thermo Fisher Scientific Cat # 22660 RNeasy minElute kit QIAGEN Cat # 74204 mMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Invitrogen RiboMinus Transcriptome Isolation Kit, yeast Thermo Fisher Scientific Cat # 10388792 QuantSeq 3’ mRNA-Seq Library Prep Kit (REV) Lexogen (Nordic Biolabs) Cat # 16.24 MAXIscript T7 transcription kit Thermo Fisher Scientific Cat # AM1312 Deposited data RNA 3’end sequencing GEO Datasets GEO: GSE218928 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Experimental models: Organisms/strains Rpb3-flag::NAT, prb1D Kind gift from John Diffley N/A HHY168, MATalpha tor1-1 fpr1::NAT RPL13A-2xFKB12::TRP1 Euroscarf Cat # Y40343 Rat1 FRB, HHY168 RAT1-FRB::KAN Baejen et al.41 N/A Ysh1 FRB, HHY168 YSH1-FRB::KAN Baejen et al.41 N/A rrp6D::His5 (s.pombe) in HHY168 this study N/A rrp6D::His5 (s.pombe) in Rat1 FRB this study N/A rrp6D::His5 (s.pombe) in Ysh1 FRB this study N/A Oligonucleotides See Table S1 for Oligonucleotides N/A Recombinant DNA pET21b Rat1-His This study pZH81 pET28a-Rai1 This study pZH82 Dual G-less cassette Genscript pZH1 Dual G-less containing yCYC1 polyA This study pZH20 Dual G-less containing ySSA1 polyA This study pZH23 pLIB Weissmann et al.74 RRID: Addgene_80610 pLIB-8His3C-spt4 This study pZH61 pLIB-spt5 This study pZH60 pYES2 Thermo Fisher Scientific Cat # V82520 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Cutadapt v2.10 Kechin et al.75 https://cutadapt.readthedocs.io/en/stable/ STAR v2.7.6a Dobin et al.76 https://github.com/alexdobin/STAR/ Samtools v1.122 Danecek et al.77 http://www.htslib.org/ deepTools v3.3.1 Ramı́rez et al.78 https://deeptools.readthedocs.io/en/develop/ BEDTools v2.27.1 Quinlan79 https://bedtools.readthedocs.io/en/latest/ Kent Tools2 Kent et al.80 N/A (Continued on next page) Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 e2 ..

    Isolation:

    Article Title: DNA-directed termination of RNA polymerase II transcription.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-RNAPII subunit Rpb1 (8WG16) Cell services, The Francis Crick Institute N/A Donkey Anti-mouse, HRP conjugate Jackson ImmunoResearch Cat # 715-035-151; RRID: AB_2340771 Donkey Anti-mouse, Alexa Fluor 680 conjugate Invitrogen Cat # A10038; RRID: AB_2534014 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat # C2988J BL21-CodonPlus (DE3)-RIPL Thermo Fisher Scientific Cat # NC9122855 DH10Bac Competent Cells Thermo Fisher Scientific Cat # 10361012 Chemicals, peptides, and recombinant proteins complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 Mono Q 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L PolyA Polymerase from E.coli NEB Cat # M0276L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L PrimeSTAR HS with GC buffer Takara Cat # R044B HisTrap FF Crude Cytiva Cat # 11000458 HiTrap Heparin HP Cytiva Cat # 17040601 Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 RNase T1 Thermo Fisher Scientific Cat # EN0541 Invitrogen 0.1 to 2kb RNA Ladder Thermo Fisher Scientific Cat # 11518766 RNA marker templates Thermo Fisher Scientific Cat # AM7782 NuPAGE 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 PVDF Membrane Merckmillipore Cat # IPFL00010 4-thiouracil Sigma-Aldrich Cat # 440736 Monoclonal ANTI-FLAG M2 antibody Sigma-Aldrich Cat # F3165-1MG (Continued on next page) e1 Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 .. REAGENT or RESOURCE SOURCE IDENTIFIER Rapamycin Sigma-Aldrich Cat # R0395 MTSEA biotin-XX linker Biotium Cat # BT90066 Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 Intercept (PBS) Blocking Buffer LI-COR Cat # 927-70001 Fluorescein-12-UTP Jena bioscience Cat # NU-821-FAMX Critical commercial assays Pierce 660nm Protein Assay Reagent Thermo Fisher Scientific Cat # 22660 RNeasy minElute kit QIAGEN Cat # 74204 mMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Invitrogen RiboMinus Transcriptome Isolation Kit, yeast Thermo Fisher Scientific Cat # 10388792 QuantSeq 3’ mRNA-Seq Library Prep Kit (REV) Lexogen (Nordic Biolabs) Cat # 16.24 MAXIscript T7 transcription kit Thermo Fisher Scientific Cat # AM1312 Deposited data RNA 3’end sequencing GEO Datasets GEO: GSE218928 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Experimental models: Organisms/strains Rpb3-flag::NAT, prb1D Kind gift from John Diffley N/A HHY168, MATalpha tor1-1 fpr1::NAT RPL13A-2xFKB12::TRP1 Euroscarf Cat # Y40343 Rat1 FRB, HHY168 RAT1-FRB::KAN Baejen et al.41 N/A Ysh1 FRB, HHY168 YSH1-FRB::KAN Baejen et al.41 N/A rrp6D::His5 (s.pombe) in HHY168 this study N/A rrp6D::His5 (s.pombe) in Rat1 FRB this study N/A rrp6D::His5 (s.pombe) in Ysh1 FRB this study N/A Oligonucleotides See Table S1 for Oligonucleotides N/A Recombinant DNA pET21b Rat1-His This study pZH81 pET28a-Rai1 This study pZH82 Dual G-less cassette Genscript pZH1 Dual G-less containing yCYC1 polyA This study pZH20 Dual G-less containing ySSA1 polyA This study pZH23 pLIB Weissmann et al.74 RRID: Addgene_80610 pLIB-8His3C-spt4 This study pZH61 pLIB-spt5 This study pZH60 pYES2 Thermo Fisher Scientific Cat # V82520 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Cutadapt v2.10 Kechin et al.75 https://cutadapt.readthedocs.io/en/stable/ STAR v2.7.6a Dobin et al.76 https://github.com/alexdobin/STAR/ Samtools v1.122 Danecek et al.77 http://www.htslib.org/ deepTools v3.3.1 Ramı́rez et al.78 https://deeptools.readthedocs.io/en/develop/ BEDTools v2.27.1 Quinlan79 https://bedtools.readthedocs.io/en/latest/ Kent Tools2 Kent et al.80 N/A (Continued on next page) Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 e2 ..

    Sequencing:

    Article Title: DNA-directed termination of RNA polymerase II transcription.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-RNAPII subunit Rpb1 (8WG16) Cell services, The Francis Crick Institute N/A Donkey Anti-mouse, HRP conjugate Jackson ImmunoResearch Cat # 715-035-151; RRID: AB_2340771 Donkey Anti-mouse, Alexa Fluor 680 conjugate Invitrogen Cat # A10038; RRID: AB_2534014 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat # C2988J BL21-CodonPlus (DE3)-RIPL Thermo Fisher Scientific Cat # NC9122855 DH10Bac Competent Cells Thermo Fisher Scientific Cat # 10361012 Chemicals, peptides, and recombinant proteins complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 Mono Q 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L PolyA Polymerase from E.coli NEB Cat # M0276L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L PrimeSTAR HS with GC buffer Takara Cat # R044B HisTrap FF Crude Cytiva Cat # 11000458 HiTrap Heparin HP Cytiva Cat # 17040601 Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 RNase T1 Thermo Fisher Scientific Cat # EN0541 Invitrogen 0.1 to 2kb RNA Ladder Thermo Fisher Scientific Cat # 11518766 RNA marker templates Thermo Fisher Scientific Cat # AM7782 NuPAGE 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 PVDF Membrane Merckmillipore Cat # IPFL00010 4-thiouracil Sigma-Aldrich Cat # 440736 Monoclonal ANTI-FLAG M2 antibody Sigma-Aldrich Cat # F3165-1MG (Continued on next page) e1 Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 .. REAGENT or RESOURCE SOURCE IDENTIFIER Rapamycin Sigma-Aldrich Cat # R0395 MTSEA biotin-XX linker Biotium Cat # BT90066 Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 Intercept (PBS) Blocking Buffer LI-COR Cat # 927-70001 Fluorescein-12-UTP Jena bioscience Cat # NU-821-FAMX Critical commercial assays Pierce 660nm Protein Assay Reagent Thermo Fisher Scientific Cat # 22660 RNeasy minElute kit QIAGEN Cat # 74204 mMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Invitrogen RiboMinus Transcriptome Isolation Kit, yeast Thermo Fisher Scientific Cat # 10388792 QuantSeq 3’ mRNA-Seq Library Prep Kit (REV) Lexogen (Nordic Biolabs) Cat # 16.24 MAXIscript T7 transcription kit Thermo Fisher Scientific Cat # AM1312 Deposited data RNA 3’end sequencing GEO Datasets GEO: GSE218928 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Experimental models: Organisms/strains Rpb3-flag::NAT, prb1D Kind gift from John Diffley N/A HHY168, MATalpha tor1-1 fpr1::NAT RPL13A-2xFKB12::TRP1 Euroscarf Cat # Y40343 Rat1 FRB, HHY168 RAT1-FRB::KAN Baejen et al.41 N/A Ysh1 FRB, HHY168 YSH1-FRB::KAN Baejen et al.41 N/A rrp6D::His5 (s.pombe) in HHY168 this study N/A rrp6D::His5 (s.pombe) in Rat1 FRB this study N/A rrp6D::His5 (s.pombe) in Ysh1 FRB this study N/A Oligonucleotides See Table S1 for Oligonucleotides N/A Recombinant DNA pET21b Rat1-His This study pZH81 pET28a-Rai1 This study pZH82 Dual G-less cassette Genscript pZH1 Dual G-less containing yCYC1 polyA This study pZH20 Dual G-less containing ySSA1 polyA This study pZH23 pLIB Weissmann et al.74 RRID: Addgene_80610 pLIB-8His3C-spt4 This study pZH61 pLIB-spt5 This study pZH60 pYES2 Thermo Fisher Scientific Cat # V82520 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Cutadapt v2.10 Kechin et al.75 https://cutadapt.readthedocs.io/en/stable/ STAR v2.7.6a Dobin et al.76 https://github.com/alexdobin/STAR/ Samtools v1.122 Danecek et al.77 http://www.htslib.org/ deepTools v3.3.1 Ramı́rez et al.78 https://deeptools.readthedocs.io/en/develop/ BEDTools v2.27.1 Quinlan79 https://bedtools.readthedocs.io/en/latest/ Kent Tools2 Kent et al.80 N/A (Continued on next page) Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 e2 ..

    Recombinant:

    Article Title: DNA-directed termination of RNA polymerase II transcription.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-RNAPII subunit Rpb1 (8WG16) Cell services, The Francis Crick Institute N/A Donkey Anti-mouse, HRP conjugate Jackson ImmunoResearch Cat # 715-035-151; RRID: AB_2340771 Donkey Anti-mouse, Alexa Fluor 680 conjugate Invitrogen Cat # A10038; RRID: AB_2534014 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat # C2988J BL21-CodonPlus (DE3)-RIPL Thermo Fisher Scientific Cat # NC9122855 DH10Bac Competent Cells Thermo Fisher Scientific Cat # 10361012 Chemicals, peptides, and recombinant proteins complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 Mono Q 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L PolyA Polymerase from E.coli NEB Cat # M0276L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L PrimeSTAR HS with GC buffer Takara Cat # R044B HisTrap FF Crude Cytiva Cat # 11000458 HiTrap Heparin HP Cytiva Cat # 17040601 Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 RNase T1 Thermo Fisher Scientific Cat # EN0541 Invitrogen 0.1 to 2kb RNA Ladder Thermo Fisher Scientific Cat # 11518766 RNA marker templates Thermo Fisher Scientific Cat # AM7782 NuPAGE 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 PVDF Membrane Merckmillipore Cat # IPFL00010 4-thiouracil Sigma-Aldrich Cat # 440736 Monoclonal ANTI-FLAG M2 antibody Sigma-Aldrich Cat # F3165-1MG (Continued on next page) e1 Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 .. REAGENT or RESOURCE SOURCE IDENTIFIER Rapamycin Sigma-Aldrich Cat # R0395 MTSEA biotin-XX linker Biotium Cat # BT90066 Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 Intercept (PBS) Blocking Buffer LI-COR Cat # 927-70001 Fluorescein-12-UTP Jena bioscience Cat # NU-821-FAMX Critical commercial assays Pierce 660nm Protein Assay Reagent Thermo Fisher Scientific Cat # 22660 RNeasy minElute kit QIAGEN Cat # 74204 mMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Invitrogen RiboMinus Transcriptome Isolation Kit, yeast Thermo Fisher Scientific Cat # 10388792 QuantSeq 3’ mRNA-Seq Library Prep Kit (REV) Lexogen (Nordic Biolabs) Cat # 16.24 MAXIscript T7 transcription kit Thermo Fisher Scientific Cat # AM1312 Deposited data RNA 3’end sequencing GEO Datasets GEO: GSE218928 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Experimental models: Organisms/strains Rpb3-flag::NAT, prb1D Kind gift from John Diffley N/A HHY168, MATalpha tor1-1 fpr1::NAT RPL13A-2xFKB12::TRP1 Euroscarf Cat # Y40343 Rat1 FRB, HHY168 RAT1-FRB::KAN Baejen et al.41 N/A Ysh1 FRB, HHY168 YSH1-FRB::KAN Baejen et al.41 N/A rrp6D::His5 (s.pombe) in HHY168 this study N/A rrp6D::His5 (s.pombe) in Rat1 FRB this study N/A rrp6D::His5 (s.pombe) in Ysh1 FRB this study N/A Oligonucleotides See Table S1 for Oligonucleotides N/A Recombinant DNA pET21b Rat1-His This study pZH81 pET28a-Rai1 This study pZH82 Dual G-less cassette Genscript pZH1 Dual G-less containing yCYC1 polyA This study pZH20 Dual G-less containing ySSA1 polyA This study pZH23 pLIB Weissmann et al.74 RRID: Addgene_80610 pLIB-8His3C-spt4 This study pZH61 pLIB-spt5 This study pZH60 pYES2 Thermo Fisher Scientific Cat # V82520 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Cutadapt v2.10 Kechin et al.75 https://cutadapt.readthedocs.io/en/stable/ STAR v2.7.6a Dobin et al.76 https://github.com/alexdobin/STAR/ Samtools v1.122 Danecek et al.77 http://www.htslib.org/ deepTools v3.3.1 Ramı́rez et al.78 https://deeptools.readthedocs.io/en/develop/ BEDTools v2.27.1 Quinlan79 https://bedtools.readthedocs.io/en/latest/ Kent Tools2 Kent et al.80 N/A (Continued on next page) Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 e2 ..

    Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat# D1916 BaseMuncher Abcam Cat# ab270049 PhosSTOP Sigma-Aldrich Cat# 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat# 11873580001 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat# 34580 Radiance plus Azure Biosystems Cat# AC2103 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Scrambled Double Serine: Bio-eahx-PP(pS)S(pS)TYPP(pS)S(pS) TYPP(pS)S(pS)TY-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2: Bio-eahx-Y(pS) PTSPSY(pS)PTSPSY(pS)PTSPS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser5: Bio-eahxYSPT(pS)PSYSPT(pS) PSYSPT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2+Ser5: Bio-eahxY(pS)PT(pS)PSY(pS)PT(pS)PSY (pS)PT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A Pierce Strep magentic beads ThermoFisher Scientific Cat# 88817 ANTI FLAG M2 agarose Sigma-Aldrich Cat# A2220 Strep-Tactin Sepharose IBA Cat# 2-1201-002 Desthiobiotin IBA Cat# 2-1000-002 Ni-NTA agarose Invitrogen Cat# R90115 Glutathione Sepharose 4B GSTtagged protein purification resin Cytiva Cat# 17075601 Amicon Ultra 4 centrifugal filter (50 kDa) Millipore Cat# UFC9050 Capto HiRes Q 5/50 Cytiva Cat# 29275878 HiTrap Desalting (5 ml) Cytiva Cat# 29048684 THZ531 MedChemExpress Cat# HY-103618 NVP-2 MedChemExpress Cat# HY-12214A Lambda Protein Phosphatase (Lambda PP) NEB Cat# P0753 FLAG-CDK12/Cyclin K This paper N/A FLAG-CDK13/Cyclin K This paper N/A FLAG-CDK9/Cyclin T1 This paper N/A GST(N)-RPB1-CTD(1586-1971) This paper N/A FLAG(N)-RPB1 This paper N/A His(N)-SSU72 This paper N/A Strep(II)(N)-PIN1 This paper N/A RNAPII from pig thymus This paper N/A FLAG(N)-CDC73 This paper N/A PAF1C This paper N/A FLAG-RTF1 This paper N/A FLAG-CDK12ΔNTD /Cyclin K This paper N/A FLAG-CDK12ΔCTD/Cyclin K This paper N/A FLAG-CDK12-KiD/Cyclin K This paper N/A FLAG-CDK12ΔRS/Cyclin K This paper N/A PAF1CΔPAF1 This paper N/A PAF1CΔCDC73 This paper N/A PAF1CΔLEO1 This paper N/A PAF1C-CDC73ΔKIM This paper N/A (Continued on next page) Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 e2

    Software:

    Article Title: DNA-directed termination of RNA polymerase II transcription.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-RNAPII subunit Rpb1 (8WG16) Cell services, The Francis Crick Institute N/A Donkey Anti-mouse, HRP conjugate Jackson ImmunoResearch Cat # 715-035-151; RRID: AB_2340771 Donkey Anti-mouse, Alexa Fluor 680 conjugate Invitrogen Cat # A10038; RRID: AB_2534014 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat # C2988J BL21-CodonPlus (DE3)-RIPL Thermo Fisher Scientific Cat # NC9122855 DH10Bac Competent Cells Thermo Fisher Scientific Cat # 10361012 Chemicals, peptides, and recombinant proteins complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat # 11873580001 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A ANTI FLAG M2 agarose Sigma-Aldrich Cat # A2220 Mono Q 5/50 GL Cytiva Cat # 17516601 Vivaspin 20 centrifugal concentrator Sigma-Aldrich Cat # Z614661 NheI-HF NEB Cat # R3131L NotI-HF NEB Cat # R3189L NcoI-HF NEB Cat # R3193L BamHI-HF NEB Cat # R3136L HindIII-HF NEB Cat # R3104L BbsI-HF NEB Cat # R3539L PolyA Polymerase from E.coli NEB Cat # M0276L fast dna ladder NEB Cat # N3238S Q5 Hot Start High-Fidelity DNA polymerase NEB Cat # M0494L PrimeSTAR HS with GC buffer Takara Cat # R044B HisTrap FF Crude Cytiva Cat # 11000458 HiTrap Heparin HP Cytiva Cat # 17040601 Superose 6 Increase 10/300 GL Cytiva Cat # 29091596 Fast Flow Q Sepharose column Cytiva Cat # 17-0510-01 MicroSpin G-25 Columns Cytiva Cat # 27532501 Grace’s Insect Medium Thermo Fisher Scientific Cat # 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat # A38915 Sf-900 III insect media Thermo Fisher Scientific Cat # 12658027 Dynabeads Myone Streptavidin T1 Thermo Fisher Scientific Cat # 65602 RNase T1 Thermo Fisher Scientific Cat # EN0541 Invitrogen 0.1 to 2kb RNA Ladder Thermo Fisher Scientific Cat # 11518766 RNA marker templates Thermo Fisher Scientific Cat # AM7782 NuPAGE 4 to 12%, Bis-Tris Thermo Fisher Scientific Cat # WG1403A Amicon Ultra-4 Centrifugal Filter Unit Merckmillipore Cat # UFC8100 PVDF Membrane Merckmillipore Cat # IPFL00010 4-thiouracil Sigma-Aldrich Cat # 440736 Monoclonal ANTI-FLAG M2 antibody Sigma-Aldrich Cat # F3165-1MG (Continued on next page) e1 Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 .. REAGENT or RESOURCE SOURCE IDENTIFIER Rapamycin Sigma-Aldrich Cat # R0395 MTSEA biotin-XX linker Biotium Cat # BT90066 Maxtract high density (200x2ml) Qiagen Cat # 129056 Micro biospin P-30 gel columns Bio-rad Cat # 732-6250 RNase OUT Invitrogen Cat # 10777-019 Intercept (PBS) Blocking Buffer LI-COR Cat # 927-70001 Fluorescein-12-UTP Jena bioscience Cat # NU-821-FAMX Critical commercial assays Pierce 660nm Protein Assay Reagent Thermo Fisher Scientific Cat # 22660 RNeasy minElute kit QIAGEN Cat # 74204 mMACS Streptavidin Kit Miltenyi Biotec Cat # 130-074-101 Invitrogen RiboMinus Transcriptome Isolation Kit, yeast Thermo Fisher Scientific Cat # 10388792 QuantSeq 3’ mRNA-Seq Library Prep Kit (REV) Lexogen (Nordic Biolabs) Cat # 16.24 MAXIscript T7 transcription kit Thermo Fisher Scientific Cat # AM1312 Deposited data RNA 3’end sequencing GEO Datasets GEO: GSE218928 Experimental models: Cell lines Sf9 Oxford Expression Technologies Cat # 600100-SF9 cells Experimental models: Organisms/strains Rpb3-flag::NAT, prb1D Kind gift from John Diffley N/A HHY168, MATalpha tor1-1 fpr1::NAT RPL13A-2xFKB12::TRP1 Euroscarf Cat # Y40343 Rat1 FRB, HHY168 RAT1-FRB::KAN Baejen et al.41 N/A Ysh1 FRB, HHY168 YSH1-FRB::KAN Baejen et al.41 N/A rrp6D::His5 (s.pombe) in HHY168 this study N/A rrp6D::His5 (s.pombe) in Rat1 FRB this study N/A rrp6D::His5 (s.pombe) in Ysh1 FRB this study N/A Oligonucleotides See Table S1 for Oligonucleotides N/A Recombinant DNA pET21b Rat1-His This study pZH81 pET28a-Rai1 This study pZH82 Dual G-less cassette Genscript pZH1 Dual G-less containing yCYC1 polyA This study pZH20 Dual G-less containing ySSA1 polyA This study pZH23 pLIB Weissmann et al.74 RRID: Addgene_80610 pLIB-8His3C-spt4 This study pZH61 pLIB-spt5 This study pZH60 pYES2 Thermo Fisher Scientific Cat # V82520 Software and algorithms ImageQuant Cytiva https://www.cytivalifesciences.com/ Cutadapt v2.10 Kechin et al.75 https://cutadapt.readthedocs.io/en/stable/ STAR v2.7.6a Dobin et al.76 https://github.com/alexdobin/STAR/ Samtools v1.122 Danecek et al.77 http://www.htslib.org/ deepTools v3.3.1 Ramı́rez et al.78 https://deeptools.readthedocs.io/en/develop/ BEDTools v2.27.1 Quinlan79 https://bedtools.readthedocs.io/en/latest/ Kent Tools2 Kent et al.80 N/A (Continued on next page) Molecular Cell 83, 3253–3267.e1–e7, September 21, 2023 e2 ..

    Produced:

    Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat# D1916 BaseMuncher Abcam Cat# ab270049 PhosSTOP Sigma-Aldrich Cat# 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat# 11873580001 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat# 34580 Radiance plus Azure Biosystems Cat# AC2103 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Scrambled Double Serine: Bio-eahx-PP(pS)S(pS)TYPP(pS)S(pS) TYPP(pS)S(pS)TY-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2: Bio-eahx-Y(pS) PTSPSY(pS)PTSPSY(pS)PTSPS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser5: Bio-eahxYSPT(pS)PSYSPT(pS) PSYSPT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2+Ser5: Bio-eahxY(pS)PT(pS)PSY(pS)PT(pS)PSY (pS)PT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A Pierce Strep magentic beads ThermoFisher Scientific Cat# 88817 ANTI FLAG M2 agarose Sigma-Aldrich Cat# A2220 Strep-Tactin Sepharose IBA Cat# 2-1201-002 Desthiobiotin IBA Cat# 2-1000-002 Ni-NTA agarose Invitrogen Cat# R90115 Glutathione Sepharose 4B GSTtagged protein purification resin Cytiva Cat# 17075601 Amicon Ultra 4 centrifugal filter (50 kDa) Millipore Cat# UFC9050 Capto HiRes Q 5/50 Cytiva Cat# 29275878 HiTrap Desalting (5 ml) Cytiva Cat# 29048684 THZ531 MedChemExpress Cat# HY-103618 NVP-2 MedChemExpress Cat# HY-12214A Lambda Protein Phosphatase (Lambda PP) NEB Cat# P0753 FLAG-CDK12/Cyclin K This paper N/A FLAG-CDK13/Cyclin K This paper N/A FLAG-CDK9/Cyclin T1 This paper N/A GST(N)-RPB1-CTD(1586-1971) This paper N/A FLAG(N)-RPB1 This paper N/A His(N)-SSU72 This paper N/A Strep(II)(N)-PIN1 This paper N/A RNAPII from pig thymus This paper N/A FLAG(N)-CDC73 This paper N/A PAF1C This paper N/A FLAG-RTF1 This paper N/A FLAG-CDK12ΔNTD /Cyclin K This paper N/A FLAG-CDK12ΔCTD/Cyclin K This paper N/A FLAG-CDK12-KiD/Cyclin K This paper N/A FLAG-CDK12ΔRS/Cyclin K This paper N/A PAF1CΔPAF1 This paper N/A PAF1CΔCDC73 This paper N/A PAF1CΔLEO1 This paper N/A PAF1C-CDC73ΔKIM This paper N/A (Continued on next page) Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 e2

    Virus:

    Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat# D1916 BaseMuncher Abcam Cat# ab270049 PhosSTOP Sigma-Aldrich Cat# 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat# 11873580001 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat# 34580 Radiance plus Azure Biosystems Cat# AC2103 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Scrambled Double Serine: Bio-eahx-PP(pS)S(pS)TYPP(pS)S(pS) TYPP(pS)S(pS)TY-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2: Bio-eahx-Y(pS) PTSPSY(pS)PTSPSY(pS)PTSPS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser5: Bio-eahxYSPT(pS)PSYSPT(pS) PSYSPT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2+Ser5: Bio-eahxY(pS)PT(pS)PSY(pS)PT(pS)PSY (pS)PT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A Pierce Strep magentic beads ThermoFisher Scientific Cat# 88817 ANTI FLAG M2 agarose Sigma-Aldrich Cat# A2220 Strep-Tactin Sepharose IBA Cat# 2-1201-002 Desthiobiotin IBA Cat# 2-1000-002 Ni-NTA agarose Invitrogen Cat# R90115 Glutathione Sepharose 4B GSTtagged protein purification resin Cytiva Cat# 17075601 Amicon Ultra 4 centrifugal filter (50 kDa) Millipore Cat# UFC9050 Capto HiRes Q 5/50 Cytiva Cat# 29275878 HiTrap Desalting (5 ml) Cytiva Cat# 29048684 THZ531 MedChemExpress Cat# HY-103618 NVP-2 MedChemExpress Cat# HY-12214A Lambda Protein Phosphatase (Lambda PP) NEB Cat# P0753 FLAG-CDK12/Cyclin K This paper N/A FLAG-CDK13/Cyclin K This paper N/A FLAG-CDK9/Cyclin T1 This paper N/A GST(N)-RPB1-CTD(1586-1971) This paper N/A FLAG(N)-RPB1 This paper N/A His(N)-SSU72 This paper N/A Strep(II)(N)-PIN1 This paper N/A RNAPII from pig thymus This paper N/A FLAG(N)-CDC73 This paper N/A PAF1C This paper N/A FLAG-RTF1 This paper N/A FLAG-CDK12ΔNTD /Cyclin K This paper N/A FLAG-CDK12ΔCTD/Cyclin K This paper N/A FLAG-CDK12-KiD/Cyclin K This paper N/A FLAG-CDK12ΔRS/Cyclin K This paper N/A PAF1CΔPAF1 This paper N/A PAF1CΔCDC73 This paper N/A PAF1CΔLEO1 This paper N/A PAF1C-CDC73ΔKIM This paper N/A (Continued on next page) Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 e2

    Transfection:

    Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat# D1916 BaseMuncher Abcam Cat# ab270049 PhosSTOP Sigma-Aldrich Cat# 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat# 11873580001 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat# 34580 Radiance plus Azure Biosystems Cat# AC2103 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Scrambled Double Serine: Bio-eahx-PP(pS)S(pS)TYPP(pS)S(pS) TYPP(pS)S(pS)TY-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2: Bio-eahx-Y(pS) PTSPSY(pS)PTSPSY(pS)PTSPS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser5: Bio-eahxYSPT(pS)PSYSPT(pS) PSYSPT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2+Ser5: Bio-eahxY(pS)PT(pS)PSY(pS)PT(pS)PSY (pS)PT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A Pierce Strep magentic beads ThermoFisher Scientific Cat# 88817 ANTI FLAG M2 agarose Sigma-Aldrich Cat# A2220 Strep-Tactin Sepharose IBA Cat# 2-1201-002 Desthiobiotin IBA Cat# 2-1000-002 Ni-NTA agarose Invitrogen Cat# R90115 Glutathione Sepharose 4B GSTtagged protein purification resin Cytiva Cat# 17075601 Amicon Ultra 4 centrifugal filter (50 kDa) Millipore Cat# UFC9050 Capto HiRes Q 5/50 Cytiva Cat# 29275878 HiTrap Desalting (5 ml) Cytiva Cat# 29048684 THZ531 MedChemExpress Cat# HY-103618 NVP-2 MedChemExpress Cat# HY-12214A Lambda Protein Phosphatase (Lambda PP) NEB Cat# P0753 FLAG-CDK12/Cyclin K This paper N/A FLAG-CDK13/Cyclin K This paper N/A FLAG-CDK9/Cyclin T1 This paper N/A GST(N)-RPB1-CTD(1586-1971) This paper N/A FLAG(N)-RPB1 This paper N/A His(N)-SSU72 This paper N/A Strep(II)(N)-PIN1 This paper N/A RNAPII from pig thymus This paper N/A FLAG(N)-CDC73 This paper N/A PAF1C This paper N/A FLAG-RTF1 This paper N/A FLAG-CDK12ΔNTD /Cyclin K This paper N/A FLAG-CDK12ΔCTD/Cyclin K This paper N/A FLAG-CDK12-KiD/Cyclin K This paper N/A FLAG-CDK12ΔRS/Cyclin K This paper N/A PAF1CΔPAF1 This paper N/A PAF1CΔCDC73 This paper N/A PAF1CΔLEO1 This paper N/A PAF1C-CDC73ΔKIM This paper N/A (Continued on next page) Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 e2

    Staining:

    Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole Sigma-Aldrich Cat# D1916 BaseMuncher Abcam Cat# ab270049 PhosSTOP Sigma-Aldrich Cat# 4906837001 complete EDTA-free protease inhibitor cocktail tablets Sigma-Aldrich Cat# 11873580001 SuperSignal West Pico PLUS Thermo Fisher Scientific Cat# 34580 Radiance plus Azure Biosystems Cat# AC2103 3xFLAG peptide Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Scrambled Double Serine: Bio-eahx-PP(pS)S(pS)TYPP(pS)S(pS) TYPP(pS)S(pS)TY-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2: Bio-eahx-Y(pS) PTSPSY(pS)PTSPSY(pS)PTSPS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser5: Bio-eahxYSPT(pS)PSYSPT(pS) PSYSPT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A CTD peptide Ser2+Ser5: Bio-eahxY(pS)PT(pS)PSY(pS)PT(pS)PSY (pS)PT(pS)PS-COOH Peptide Chemistry, The Francis Crick Institute N/A Pierce Strep magentic beads ThermoFisher Scientific Cat# 88817 ANTI FLAG M2 agarose Sigma-Aldrich Cat# A2220 Strep-Tactin Sepharose IBA Cat# 2-1201-002 Desthiobiotin IBA Cat# 2-1000-002 Ni-NTA agarose Invitrogen Cat# R90115 Glutathione Sepharose 4B GSTtagged protein purification resin Cytiva Cat# 17075601 Amicon Ultra 4 centrifugal filter (50 kDa) Millipore Cat# UFC9050 Capto HiRes Q 5/50 Cytiva Cat# 29275878 HiTrap Desalting (5 ml) Cytiva Cat# 29048684 THZ531 MedChemExpress Cat# HY-103618 NVP-2 MedChemExpress Cat# HY-12214A Lambda Protein Phosphatase (Lambda PP) NEB Cat# P0753 FLAG-CDK12/Cyclin K This paper N/A FLAG-CDK13/Cyclin K This paper N/A FLAG-CDK9/Cyclin T1 This paper N/A GST(N)-RPB1-CTD(1586-1971) This paper N/A FLAG(N)-RPB1 This paper N/A His(N)-SSU72 This paper N/A Strep(II)(N)-PIN1 This paper N/A RNAPII from pig thymus This paper N/A FLAG(N)-CDC73 This paper N/A PAF1C This paper N/A FLAG-RTF1 This paper N/A FLAG-CDK12ΔNTD /Cyclin K This paper N/A FLAG-CDK12ΔCTD/Cyclin K This paper N/A FLAG-CDK12-KiD/Cyclin K This paper N/A FLAG-CDK12ΔRS/Cyclin K This paper N/A PAF1CΔPAF1 This paper N/A PAF1CΔCDC73 This paper N/A PAF1CΔLEO1 This paper N/A PAF1C-CDC73ΔKIM This paper N/A (Continued on next page) Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025 e2



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