Virus:Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-STAG1 Abcam Cat# ab4457; RRID: AB_2286589 anti-STAG2 Santa Cruz Cat# sc-81852; RRID: AB_2199948 anti-STAG2 Abcam Cat# ab4463; RRID: AB_304471 anti-FLI1 Abcam Cat# ab133485; RRID: AB_2722650 anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 anti-H3 Abcam Cat# ab1791; RRID: AB_302613 anti-beta-Actin anti-beta-Actin Cat# A5316; RRID: AB_476743 anti-rabbit-IgG-HRP GE Healthcare Cat# NA934; RRID: AB_772206 anti-mouse-IgG-HRP GE Healthcare Cat# NXA931; RRID: AB_772209 anti-FLI1 Abcam Cat# ab15289; RRID: AB_301825 anti-RAD21 Abcam Cat# ab992; RRID: AB_2176601 anti-CTCF Diagenode Cat# C15410210; RRID: AB_2753160 anti-SMC1 Bethyl laboratories Cat# A300-055A; RRID: AB_2192467 anti-H3K4me3 Diagenode Cat# pAb-003-050; RRID: AB_2616052 Anti-paxillin BD biosciences Cat# 610051; RRID: AB_397463 Phalloidin-tetramethylrhodamine B isothiocyanate Sigma Cat# P1951; RRID: AB_2315148 anti-mouse-IgG-Alexa Fluor 488 Invitrogen Cat# A-21202; RRID: AB_141607 Bacterial and virus strains MLM3636 vector Addgene Addgene_43860 pCas9_GFP Addgene Addgene_44719 pCDH1-CMV-MCS-EF1-Puro System Biosciences, CA CD510B-1 Stellar competent cells Ozyme 636763 Biological samples Frozen human tumor samples Curie Institute, UGS N/A Chemicals, peptides, and recombinant proteins Alpha-MEM Gibco 22561021 DMEM GE Healthcare SH30022.01 IMDM Gibco 12440-53 RPMI GE Healthcare SH30027.01 fetal bovine serum GE Helthcare SV30160.03 MSC qualified fetal bovine serum Gibco 2662029 Insuline-Transferrin-selenium 1X Gibco 41400-045 10X T4 ligation buffer New England Biolabs B0202S PNK Fermentas EK0031 10X Tango buffer Fermentas BY5 DTT Sigma D0632 ATP Promega U120D low melting point agarose Invitrogen 16520 Ultra-Low Attachment microplates Corning 7007 Collagen I, High Concentration, Rat Tail, Corning 354249 BsmB1 New England Biolabs R05805 T4 DNA Ligase New England Biolabs M0202S 4-15% Mini-PROTEAN TGX gels BIO-RAD 456-1084 (Continued on next page) e1 Cancer Cell 39, 1–17.e1–e9, June 14, 2021 .. REAGENT or RESOURCE SOURCE IDENTIFIER nitrocellulose membranes BIO-RAD 1704159 SuperSignal West Pico Plus Thermo Scientific 34580 methanol-free formaldehyde Thermo-Scientific 28908 protease inhibitor cocktail Roche 11836145001 Mbo1 New England Biolabs R0147M TN5 Illumina 15028211 phusion HF 2X New England Biolabs M0531S Evagreen 20X Biotium 31000 Ampure XP beads Beckman Coulter A63881 Critical commercial assays mycoplasma detection kit Minerva Biolabs 11-9250 QiAamp DNA mini kit Qiagen 51304 Nucleofast 96 PCR plate Macherey Nagel 743100.50 RNeasy plus mini kit Qiagen 74134 Lipofectamine RNAiMAX Invitrogen 13778030 cDNA Reverse Transcription Kit Applied Biosystems 4368814 Power SYBR Green PCR Master Applied Biosystems 4367659 TruSeq Stranded mRNA Library preparation kit Illumina 20020594 iDeal ChIP-seq kit for transcription factors Diagenode C01010170 iDeal ChIP-seq kit for for histones Diagenode C01010171 TruSeq ChIP library preparation kit Illumina IP-202-1012 Deposited data Ewing sarcoma RNAseq, ChiP-Seq, HiChIP, Affymetrix arrays This paper GSE133228 Ewing sarcoma transcriptome Tirode et al., 2014 EGAS00001003333 Ewing sarcoma Affymetrix arrays Postel-Vinay et al., 2012 GSE34620 Experimental models: cell lines
Plasmid Preparation:Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-STAG1 Abcam Cat# ab4457; RRID: AB_2286589 anti-STAG2 Santa Cruz Cat# sc-81852; RRID: AB_2199948 anti-STAG2 Abcam Cat# ab4463; RRID: AB_304471 anti-FLI1 Abcam Cat# ab133485; RRID: AB_2722650 anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 anti-H3 Abcam Cat# ab1791; RRID: AB_302613 anti-beta-Actin anti-beta-Actin Cat# A5316; RRID: AB_476743 anti-rabbit-IgG-HRP GE Healthcare Cat# NA934; RRID: AB_772206 anti-mouse-IgG-HRP GE Healthcare Cat# NXA931; RRID: AB_772209 anti-FLI1 Abcam Cat# ab15289; RRID: AB_301825 anti-RAD21 Abcam Cat# ab992; RRID: AB_2176601 anti-CTCF Diagenode Cat# C15410210; RRID: AB_2753160 anti-SMC1 Bethyl laboratories Cat# A300-055A; RRID: AB_2192467 anti-H3K4me3 Diagenode Cat# pAb-003-050; RRID: AB_2616052 Anti-paxillin BD biosciences Cat# 610051; RRID: AB_397463 Phalloidin-tetramethylrhodamine B isothiocyanate Sigma Cat# P1951; RRID: AB_2315148 anti-mouse-IgG-Alexa Fluor 488 Invitrogen Cat# A-21202; RRID: AB_141607 Bacterial and virus strains MLM3636 vector Addgene Addgene_43860 pCas9_GFP Addgene Addgene_44719 pCDH1-CMV-MCS-EF1-Puro System Biosciences, CA CD510B-1 Stellar competent cells Ozyme 636763 Biological samples Frozen human tumor samples Curie Institute, UGS N/A Chemicals, peptides, and recombinant proteins Alpha-MEM Gibco 22561021 DMEM GE Healthcare SH30022.01 IMDM Gibco 12440-53 RPMI GE Healthcare SH30027.01 fetal bovine serum GE Helthcare SV30160.03 MSC qualified fetal bovine serum Gibco 2662029 Insuline-Transferrin-selenium 1X Gibco 41400-045 10X T4 ligation buffer New England Biolabs B0202S PNK Fermentas EK0031 10X Tango buffer Fermentas BY5 DTT Sigma D0632 ATP Promega U120D low melting point agarose Invitrogen 16520 Ultra-Low Attachment microplates Corning 7007 Collagen I, High Concentration, Rat Tail, Corning 354249 BsmB1 New England Biolabs R05805 T4 DNA Ligase New England Biolabs M0202S 4-15% Mini-PROTEAN TGX gels BIO-RAD 456-1084 (Continued on next page) e1 Cancer Cell 39, 1–17.e1–e9, June 14, 2021 .. REAGENT or RESOURCE SOURCE IDENTIFIER nitrocellulose membranes BIO-RAD 1704159 SuperSignal West Pico Plus Thermo Scientific 34580 methanol-free formaldehyde Thermo-Scientific 28908 protease inhibitor cocktail Roche 11836145001 Mbo1 New England Biolabs R0147M TN5 Illumina 15028211 phusion HF 2X New England Biolabs M0531S Evagreen 20X Biotium 31000 Ampure XP beads Beckman Coulter A63881 Critical commercial assays mycoplasma detection kit Minerva Biolabs 11-9250 QiAamp DNA mini kit Qiagen 51304 Nucleofast 96 PCR plate Macherey Nagel 743100.50 RNeasy plus mini kit Qiagen 74134 Lipofectamine RNAiMAX Invitrogen 13778030 cDNA Reverse Transcription Kit Applied Biosystems 4368814 Power SYBR Green PCR Master Applied Biosystems 4367659 TruSeq Stranded mRNA Library preparation kit Illumina 20020594 iDeal ChIP-seq kit for transcription factors Diagenode C01010170 iDeal ChIP-seq kit for for histones Diagenode C01010171 TruSeq ChIP library preparation kit Illumina IP-202-1012 Deposited data Ewing sarcoma RNAseq, ChiP-Seq, HiChIP, Affymetrix arrays This paper GSE133228 Ewing sarcoma transcriptome Tirode et al., 2014 EGAS00001003333 Ewing sarcoma Affymetrix arrays Postel-Vinay et al., 2012 GSE34620 Experimental models: cell lines
Recombinant:Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-STAG1 Abcam Cat# ab4457; RRID: AB_2286589 anti-STAG2 Santa Cruz Cat# sc-81852; RRID: AB_2199948 anti-STAG2 Abcam Cat# ab4463; RRID: AB_304471 anti-FLI1 Abcam Cat# ab133485; RRID: AB_2722650 anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 anti-H3 Abcam Cat# ab1791; RRID: AB_302613 anti-beta-Actin anti-beta-Actin Cat# A5316; RRID: AB_476743 anti-rabbit-IgG-HRP GE Healthcare Cat# NA934; RRID: AB_772206 anti-mouse-IgG-HRP GE Healthcare Cat# NXA931; RRID: AB_772209 anti-FLI1 Abcam Cat# ab15289; RRID: AB_301825 anti-RAD21 Abcam Cat# ab992; RRID: AB_2176601 anti-CTCF Diagenode Cat# C15410210; RRID: AB_2753160 anti-SMC1 Bethyl laboratories Cat# A300-055A; RRID: AB_2192467 anti-H3K4me3 Diagenode Cat# pAb-003-050; RRID: AB_2616052 Anti-paxillin BD biosciences Cat# 610051; RRID: AB_397463 Phalloidin-tetramethylrhodamine B isothiocyanate Sigma Cat# P1951; RRID: AB_2315148 anti-mouse-IgG-Alexa Fluor 488 Invitrogen Cat# A-21202; RRID: AB_141607 Bacterial and virus strains MLM3636 vector Addgene Addgene_43860 pCas9_GFP Addgene Addgene_44719 pCDH1-CMV-MCS-EF1-Puro System Biosciences, CA CD510B-1 Stellar competent cells Ozyme 636763 Biological samples Frozen human tumor samples Curie Institute, UGS N/A Chemicals, peptides, and recombinant proteins Alpha-MEM Gibco 22561021 DMEM GE Healthcare SH30022.01 IMDM Gibco 12440-53 RPMI GE Healthcare SH30027.01 fetal bovine serum GE Helthcare SV30160.03 MSC qualified fetal bovine serum Gibco 2662029 Insuline-Transferrin-selenium 1X Gibco 41400-045 10X T4 ligation buffer New England Biolabs B0202S PNK Fermentas EK0031 10X Tango buffer Fermentas BY5 DTT Sigma D0632 ATP Promega U120D low melting point agarose Invitrogen 16520 Ultra-Low Attachment microplates Corning 7007 Collagen I, High Concentration, Rat Tail, Corning 354249 BsmB1 New England Biolabs R05805 T4 DNA Ligase New England Biolabs M0202S 4-15% Mini-PROTEAN TGX gels BIO-RAD 456-1084 (Continued on next page) e1 Cancer Cell 39, 1–17.e1–e9, June 14, 2021 .. REAGENT or RESOURCE SOURCE IDENTIFIER nitrocellulose membranes BIO-RAD 1704159 SuperSignal West Pico Plus Thermo Scientific 34580 methanol-free formaldehyde Thermo-Scientific 28908 protease inhibitor cocktail Roche 11836145001 Mbo1 New England Biolabs R0147M TN5 Illumina 15028211 phusion HF 2X New England Biolabs M0531S Evagreen 20X Biotium 31000 Ampure XP beads Beckman Coulter A63881 Critical commercial assays mycoplasma detection kit Minerva Biolabs 11-9250 QiAamp DNA mini kit Qiagen 51304 Nucleofast 96 PCR plate Macherey Nagel 743100.50 RNeasy plus mini kit Qiagen 74134 Lipofectamine RNAiMAX Invitrogen 13778030 cDNA Reverse Transcription Kit Applied Biosystems 4368814 Power SYBR Green PCR Master Applied Biosystems 4367659 TruSeq Stranded mRNA Library preparation kit Illumina 20020594 iDeal ChIP-seq kit for transcription factors Diagenode C01010170 iDeal ChIP-seq kit for for histones Diagenode C01010171 TruSeq ChIP library preparation kit Illumina IP-202-1012 Deposited data Ewing sarcoma RNAseq, ChiP-Seq, HiChIP, Affymetrix arrays This paper GSE133228 Ewing sarcoma transcriptome Tirode et al., 2014 EGAS00001003333 Ewing sarcoma Affymetrix arrays Postel-Vinay et al., 2012 GSE34620 Experimental models: cell lines
Article Title: Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RAD51 primary antibody (rabbit) BioAcademia Cat# 70-002 53BP1 primary antibody (rabbit) Novus Biologicals Cat# NB100-304; RRID:AB_10003037 53BP1 primary antibody (mouse) Thanos Halazonetis (Schultz et al., 2000) N/A H2AX Phospho S139 primary antibody (rabbit) Cell Signaling Cat# 9718; RRID:AB_2118009 Histone H3 Phospho S10 primary antibody (mouse) Cell Signaling Cat# 9701; RRID:AB_331535 BRCA2 primary antibody (mouse) Calbiochem Cat# OP95; RRID:AB_2067762 SMC1 primary antibody (rabbit) Bethyl Laboratories Cat# A300-055A; RRID:AB_2192467 GAPDH primary antibody (mouse) Novus Biologicals Cat# NB600-502; RRID:AB_10077682 FLAG primary antibody (mouse) Agilent Technologies (Stratagene) Cat# 200470-21 KAP1 Phospho S824 primary antibody (rabbit) Bethyl Laboratories Cat# A300-767A; RRID:AB_669740 KAP1 (rabbit) Bethyl Laboratories Cat# A300-274A; RRID:AB_185559 Alexa Fluor 568 goat anti-rabbit ThermoFisher Cat# A11036; RRID:AB_10563566 Alexa Fluor 594 goat anti-rabbit ThermoFisher Cat # A11012; RRID:AB_2534079 Alexa Fluor 488 goat anti-mouse ThermoFisher Cat# A11029; RRID:AB_2534088 Alexa Fluor 488 goat anti-mouse ThermoFisher Cat# A10667; RRID:AB_2534057 anti-mouse HRP-conjugated secondary antibody DAKO Cat# P0447; RRID:AB_2617137 anti-rabbit HRP-conjugated secondary antibody DAKO Cat# P0448; RRID:AB_2617138 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Sigma-Aldrich Cat# D5796 Tet system approved fetal bovine serum Takara Bio Cat# 631106 Doxycycline Sigma-Aldrich Cat# D9891 Tetracycline Sigma-Aldrich Cat# T7660 Thymidine Sigma-Aldrich Cat# T1895 RO-3306 Sigma-Aldrich Cat# SML0569 Hydroxyurea Sigma-Aldrich Cat# H8627 Nocodazole Sigma-Aldrich Cat# M1404 EdU ThermoFisher Cat# C10340 Aphidicolin Sigma-Aldrich Cat# A0781 DRB Sigma-Aldrich Cat# D1916 EU ThermoFisher Cat# E10345 INTERFERin (transfection reagent) Polyplus Cat# 409-01 Dharmafect1 (transfection reagent) Horizon Discovery Cat# T-2001-03 Azide-PEG(3+3)-S-S-biotin Jena Biosciences Cat# CLK-A2112-10 Dynabeads MyOne streptavidin C1 ThermoFisher Cat# 65001 b-mercaptoethanol Sigma-Aldrich Cat# M6250 TRIzol reagent ThermoFisher Cat# 15596026 HiMark Pre-stained Protein Standard ThermoFisher Cat# LC5699 (Continued on next page) e1 Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 .. REAGENT or RESOURCE SOURCE IDENTIFIER ProLong Gold Antifade Mountant ThermoFisher Cat# P36930 DAPI ThermoFisher Cat# D1306 Mowiol 4-88 Sigma-Aldrich Cat# 475904 Propidium iodide solution Sigma-Aldrich Cat# P4864 PureLink RNase A ThermoFisher Cat# 12091021 Critical commercial assays Click-iT EdU Alexa Fluor 647 Cell Proliferation Kit for Imaging ThermoFisher Cat# C10340 Click-iT Nascent RNA Capture Kit ThermoFisher Cat# C10365 Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit ThermoFisher Cat# C10424 TruSeq ChIP Library Preparation Kit Illumina Cat# IP-202-1012 TruSeq Stranded Total RNA with RiboZero Gold Illumina Cat# RS-122-2301 Deposited data DRIP-seq data (HeLa cells) Hamperl et al., 2017 GEO: GSM2452072 qDRIP-seq data (HeLa cells) Crossley et al., 2020 GEO: GSE134084 OK-seq data (RFD in HeLa cells) Petryk et al., 2016 https://github.com/CL-CHEN-Lab/OK- Seq/tree/master/published_results/HeLa EdU-seq BRCA2-deficient H1299 Dagg et al., 2021 GEO: GSM4650337 MiDAS-seq data This paper GEO: GSE196163 EU-seq data This paper GEO: GSE196163 Experimental models: Cell lines H1299 +shBRCA2DOX cells (male origin) Zimmer et al., 2016 N/A HeLa cells (female origin) ATCC Cat# CCL-2 HeLa +RNaseH1-FLAGDOX (female origin) Sollier et al., 2014 N/A U2OS CyclinE (tet OFF) RAD52+/+ (female origin) Sotiriou et al., 2016 N/A U2OS CyclinE (tet OFF) RAD52-/- (female origin) Sotiriou et al., 2016 N/A U2OS T-REx GFP-RnaseH1D210N (female origin) Teloni et al., 2019 N/A Oligonucleotides siGENOME BRCA2 siRNA (SMARTpool) Horizon Discovery Cat# M-003462-01 ON-TARGETplus RAD52 siRNA (SMARTpool) Horizon Discovery Cat# L-011760-00-0005 BRCA2 siRNA Qiagen Cat# SI02653434 AllStar negative control Qiagen Cat# 1027281 TP53 siRNA Dharmacon Cat# GEHCU-000146 ON-TARGETplus non-targeting control pool Horizon Discovery Cat# D-001810-10-05 Software and algorithms GraphPad Prism GraphPad https://www.graphpad.com/ Olympus ScanR Image Analysis Software 3.0.0 Olympus https://www.olympus-lifescience.com/en/ microscopes/inverted/scanr/ TIBCO Spotfire TIBCO Software https://www.tibco.com/products/tibco- spotfire Fiji Schindelin et al., 2012 https://fiji.sc/ Bedtools Quinlan and Hall, 2010 https://bedtools.readthedocs.io/en/latest/ (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 e2
Article Title: Perturbations in 3D genome organization can promote acquired drug resistance.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies H3k27me3 Actif Motif Cat# #39155; RRID:AB_2561020 H3k9me2 Abcam Cat# ab1220; RRID:AB_449854 Lamin B1 Abcam Cat# ab16048; RRID:AB_443298 Goat anti-rabbit Alexa Fluor 488-conjugated secondary antibody Invitrogen; Thermo Fisher Scientific Cat# A11008; RRID:AB_143165 Goat anti-mouse Alexa Fluor 568-conjugated secondary antibody Invitrogen; Thermo Fisher Scientific Cat# A11004; RRID:AB_253407 SMC1 Bethyl Cat# A300-055A; RRID:AB_2192467 Alpha-tubulin Sigma-Aldrich Cat# T5168; RRID:AB_477579 DNMT1 Sigma-Aldrich Cat# D4692; RRID:AB_262096 H3k27ac Actif Motif Cat# 39133; AB_2722569 Lamin A/C Santa Cruz Cat# sc6215; RRID:AB_648152 Lamin B Receptor (LBR) Abcam Cat# ab122919; RRID:AB_10902156 Polyclonal Goat Anti-Rabbit Immunoglobulins/HRP Dako Cat# P448; RRID:AB_2617138 Polyclonal Goat Anti-Mouse Immunoglobulins/HRP Dako Cat# P447; RRID:AB_2617137 Polyclonal Rabbit Anti-Goat Immunoglobulins/HRP Dako Cat# P449; RRID:AB_2617143 H3k9me3 Abcam Cat# ab8898; RRID:AB_306848 H2AZ Abcam Cat# ab4174; RRID:AB_30434 H3k4me1 Abcam Cat# ab8895; RRID:AB_306847 H3k4me3 Abcam Cat# ab8580; RRID:AB_306649 H3k36me3 Abcam Cat# ab9050; RRID:AB_306966 Lamin B2 Abcam Cat# ab8983; RRID:AB_306912 Chemicals, peptides, and recombinant proteins Dimethyl sulfoxide (DMSO) Sigma-Aldrich Cat# D8418 DMEM/F-12 Gibco Cat# 11320033 RPMI 1640 Gibco Cat#: 21875034 DMEM Gibco Cat# 11965092 Fetal Bovine Serum Serana Cat# S-FBS-EU-015 Penicillin-Streptomycin Gibco Cat# 15070063 Sodium Piruvate Gibco Cat# 11360070 (Continued on next page) Cell Reports 42, 113124, October 31, 2023 17 .. REAGENT or RESOURCE SOURCE IDENTIFIER HEPES Gibco Cat# 15630080 Prolong Gold Antifade Mountant Invitrogen; Thermo Fisher Scientific Cat# P36934 Lipofectamine RNAiMAX Thermo Fisher Scientific Cat# 13778150 M3814 (DNAPKi) Selleckchem Cat# S8586 Gibson Assembly Master Mix Biolabs Cat# E2611S Taxol (Paclitaxel) Sigma-Aldrich Cat# 33069-62-4 5-Aza-2-deoxycytidine Sigma-Aldrich Cat# A3656 GSK126 Selleckchem Cat# S7061 BIX-01294 Selleckchem Cat# S8006 NEB 5x Taq master mix Biolabs Cat# M0285L Phusion High-Fidelity DNA Polymerase Biolabs Cat# M0530S SSC Buffer 20x Concentrate Sigma-Aldrich Cat# S6639 Formamide (Deionized) Thermo Fisher Scientific Cat# AM9342 Fugene 6 Promega Cat# E5911 DynabeadsTM Protein A Thermo Fisher Scientific Cat# 10001D SuperScriptTM II Reverse Transcriptase Thermo Fisher Scientific Cat# 18064014 Critical commercial assays RNeasy Mini Kit Qiagen Cat# 74104 BioScriptTM Reverse Transcriptase Bioline Cat# BIO-27036 PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 QIAquick Gel Extraction Kit Qiagen Cat# 28704 Dual-Luciferase Reporter Assay System Kit Promega Cat# E1910 ISOLATE II Genomic DNA Kit Meridian Bioscience Cat #BIO-52067 EpiJET Bisulfite Conversion Kit Thermo Fisher Scientific Cat# K1461 MyTaq Red HS Mix Meridian Bioscience Cat# BIO-25046 Experimental models: Cell lines RPE-hTERT iCut van den Berg et al.56 N/A RPE-hTERT CRISPRa Brueckner et al.52 N/A A549 NKI Cryostorage N/A MDA-MB-231 NKI Cryostorage N/A FADU NKI Cryostorage N/A Oligonucleotides sgRNAs for CRISPRa, see Table S1 This paper N/A crRNAs for RPE iCUT KOs, see Table S2 This paper N/A RT-qPCR and ChIP-qPCR primers, see Table S3 This paper N/A RT-qPCR primers position, see Table S4 This paper N/A Recombinant DNA ANCHOR3 plasmids NeoVirTech N/A pUC19 plasmid Addgene Cat# 49793 pSpCas9(BB)-2A-Puro (PX459) Addgene Cat# 62988 pSpCas9(BB)-2A-Puro-ABCB1 This paper N/A pGL3-Basic Vector GenBank Addgene GenBank: U47295 pGL3-Promoter Vector GenBank Addgene GeneBank: U47298 pRL-SV40 Vector GenBank Addgene GeneBank: AF025845 pGL3-ABCB1 This paper N/A (Continued on next page) 18 Cell Reports 42, 113124, October 31, 2023
Ligation:Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-STAG1 Abcam Cat# ab4457; RRID: AB_2286589 anti-STAG2 Santa Cruz Cat# sc-81852; RRID: AB_2199948 anti-STAG2 Abcam Cat# ab4463; RRID: AB_304471 anti-FLI1 Abcam Cat# ab133485; RRID: AB_2722650 anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 anti-H3 Abcam Cat# ab1791; RRID: AB_302613 anti-beta-Actin anti-beta-Actin Cat# A5316; RRID: AB_476743 anti-rabbit-IgG-HRP GE Healthcare Cat# NA934; RRID: AB_772206 anti-mouse-IgG-HRP GE Healthcare Cat# NXA931; RRID: AB_772209 anti-FLI1 Abcam Cat# ab15289; RRID: AB_301825 anti-RAD21 Abcam Cat# ab992; RRID: AB_2176601 anti-CTCF Diagenode Cat# C15410210; RRID: AB_2753160 anti-SMC1 Bethyl laboratories Cat# A300-055A; RRID: AB_2192467 anti-H3K4me3 Diagenode Cat# pAb-003-050; RRID: AB_2616052 Anti-paxillin BD biosciences Cat# 610051; RRID: AB_397463 Phalloidin-tetramethylrhodamine B isothiocyanate Sigma Cat# P1951; RRID: AB_2315148 anti-mouse-IgG-Alexa Fluor 488 Invitrogen Cat# A-21202; RRID: AB_141607 Bacterial and virus strains MLM3636 vector Addgene Addgene_43860 pCas9_GFP Addgene Addgene_44719 pCDH1-CMV-MCS-EF1-Puro System Biosciences, CA CD510B-1 Stellar competent cells Ozyme 636763 Biological samples Frozen human tumor samples Curie Institute, UGS N/A Chemicals, peptides, and recombinant proteins Alpha-MEM Gibco 22561021 DMEM GE Healthcare SH30022.01 IMDM Gibco 12440-53 RPMI GE Healthcare SH30027.01 fetal bovine serum GE Helthcare SV30160.03 MSC qualified fetal bovine serum Gibco 2662029 Insuline-Transferrin-selenium 1X Gibco 41400-045 10X T4 ligation buffer New England Biolabs B0202S PNK Fermentas EK0031 10X Tango buffer Fermentas BY5 DTT Sigma D0632 ATP Promega U120D low melting point agarose Invitrogen 16520 Ultra-Low Attachment microplates Corning 7007 Collagen I, High Concentration, Rat Tail, Corning 354249 BsmB1 New England Biolabs R05805 T4 DNA Ligase New England Biolabs M0202S 4-15% Mini-PROTEAN TGX gels BIO-RAD 456-1084 (Continued on next page) e1 Cancer Cell 39, 1–17.e1–e9, June 14, 2021 .. REAGENT or RESOURCE SOURCE IDENTIFIER nitrocellulose membranes BIO-RAD 1704159 SuperSignal West Pico Plus Thermo Scientific 34580 methanol-free formaldehyde Thermo-Scientific 28908 protease inhibitor cocktail Roche 11836145001 Mbo1 New England Biolabs R0147M TN5 Illumina 15028211 phusion HF 2X New England Biolabs M0531S Evagreen 20X Biotium 31000 Ampure XP beads Beckman Coulter A63881 Critical commercial assays mycoplasma detection kit Minerva Biolabs 11-9250 QiAamp DNA mini kit Qiagen 51304 Nucleofast 96 PCR plate Macherey Nagel 743100.50 RNeasy plus mini kit Qiagen 74134 Lipofectamine RNAiMAX Invitrogen 13778030 cDNA Reverse Transcription Kit Applied Biosystems 4368814 Power SYBR Green PCR Master Applied Biosystems 4367659 TruSeq Stranded mRNA Library preparation kit Illumina 20020594 iDeal ChIP-seq kit for transcription factors Diagenode C01010170 iDeal ChIP-seq kit for for histones Diagenode C01010171 TruSeq ChIP library preparation kit Illumina IP-202-1012 Deposited data Ewing sarcoma RNAseq, ChiP-Seq, HiChIP, Affymetrix arrays This paper GSE133228 Ewing sarcoma transcriptome Tirode et al., 2014 EGAS00001003333 Ewing sarcoma Affymetrix arrays Postel-Vinay et al., 2012 GSE34620 Experimental models: cell lines
Concentration Assay:Article Title: STAG2 mutations alter CTCF-anchored loop extrusion, reduce cis-regulatory interactions and EWSR1-FLI1 activity in Ewing sarcoma.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-STAG1 Abcam Cat# ab4457; RRID: AB_2286589 anti-STAG2 Santa Cruz Cat# sc-81852; RRID: AB_2199948 anti-STAG2 Abcam Cat# ab4463; RRID: AB_304471 anti-FLI1 Abcam Cat# ab133485; RRID: AB_2722650 anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 anti-H3 Abcam Cat# ab1791; RRID: AB_302613 anti-beta-Actin anti-beta-Actin Cat# A5316; RRID: AB_476743 anti-rabbit-IgG-HRP GE Healthcare Cat# NA934; RRID: AB_772206 anti-mouse-IgG-HRP GE Healthcare Cat# NXA931; RRID: AB_772209 anti-FLI1 Abcam Cat# ab15289; RRID: AB_301825 anti-RAD21 Abcam Cat# ab992; RRID: AB_2176601 anti-CTCF Diagenode Cat# C15410210; RRID: AB_2753160 anti-SMC1 Bethyl laboratories Cat# A300-055A; RRID: AB_2192467 anti-H3K4me3 Diagenode Cat# pAb-003-050; RRID: AB_2616052 Anti-paxillin BD biosciences Cat# 610051; RRID: AB_397463 Phalloidin-tetramethylrhodamine B isothiocyanate Sigma Cat# P1951; RRID: AB_2315148 anti-mouse-IgG-Alexa Fluor 488 Invitrogen Cat# A-21202; RRID: AB_141607 Bacterial and virus strains MLM3636 vector Addgene Addgene_43860 pCas9_GFP Addgene Addgene_44719 pCDH1-CMV-MCS-EF1-Puro System Biosciences, CA CD510B-1 Stellar competent cells Ozyme 636763 Biological samples Frozen human tumor samples Curie Institute, UGS N/A Chemicals, peptides, and recombinant proteins Alpha-MEM Gibco 22561021 DMEM GE Healthcare SH30022.01 IMDM Gibco 12440-53 RPMI GE Healthcare SH30027.01 fetal bovine serum GE Helthcare SV30160.03 MSC qualified fetal bovine serum Gibco 2662029 Insuline-Transferrin-selenium 1X Gibco 41400-045 10X T4 ligation buffer New England Biolabs B0202S PNK Fermentas EK0031 10X Tango buffer Fermentas BY5 DTT Sigma D0632 ATP Promega U120D low melting point agarose Invitrogen 16520 Ultra-Low Attachment microplates Corning 7007 Collagen I, High Concentration, Rat Tail, Corning 354249 BsmB1 New England Biolabs R05805 T4 DNA Ligase New England Biolabs M0202S 4-15% Mini-PROTEAN TGX gels BIO-RAD 456-1084 (Continued on next page) e1 Cancer Cell 39, 1–17.e1–e9, June 14, 2021 .. REAGENT or RESOURCE SOURCE IDENTIFIER nitrocellulose membranes BIO-RAD 1704159 SuperSignal West Pico Plus Thermo Scientific 34580 methanol-free formaldehyde Thermo-Scientific 28908 protease inhibitor cocktail Roche 11836145001 Mbo1 New England Biolabs R0147M TN5 Illumina 15028211 phusion HF 2X New England Biolabs M0531S Evagreen 20X Biotium 31000 Ampure XP beads Beckman Coulter A63881 Critical commercial assays mycoplasma detection kit Minerva Biolabs 11-9250 QiAamp DNA mini kit Qiagen 51304 Nucleofast 96 PCR plate Macherey Nagel 743100.50 RNeasy plus mini kit Qiagen 74134 Lipofectamine RNAiMAX Invitrogen 13778030 cDNA Reverse Transcription Kit Applied Biosystems 4368814 Power SYBR Green PCR Master Applied Biosystems 4367659 TruSeq Stranded mRNA Library preparation kit Illumina 20020594 iDeal ChIP-seq kit for transcription factors Diagenode C01010170 iDeal ChIP-seq kit for for histones Diagenode C01010171 TruSeq ChIP library preparation kit Illumina IP-202-1012 Deposited data Ewing sarcoma RNAseq, ChiP-Seq, HiChIP, Affymetrix arrays This paper GSE133228 Ewing sarcoma transcriptome Tirode et al., 2014 EGAS00001003333 Ewing sarcoma Affymetrix arrays Postel-Vinay et al., 2012 GSE34620 Experimental models: cell lines
Modification:Article Title: Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RAD51 primary antibody (rabbit) BioAcademia Cat# 70-002 53BP1 primary antibody (rabbit) Novus Biologicals Cat# NB100-304; RRID:AB_10003037 53BP1 primary antibody (mouse) Thanos Halazonetis (Schultz et al., 2000) N/A H2AX Phospho S139 primary antibody (rabbit) Cell Signaling Cat# 9718; RRID:AB_2118009 Histone H3 Phospho S10 primary antibody (mouse) Cell Signaling Cat# 9701; RRID:AB_331535 BRCA2 primary antibody (mouse) Calbiochem Cat# OP95; RRID:AB_2067762 SMC1 primary antibody (rabbit) Bethyl Laboratories Cat# A300-055A; RRID:AB_2192467 GAPDH primary antibody (mouse) Novus Biologicals Cat# NB600-502; RRID:AB_10077682 FLAG primary antibody (mouse) Agilent Technologies (Stratagene) Cat# 200470-21 KAP1 Phospho S824 primary antibody (rabbit) Bethyl Laboratories Cat# A300-767A; RRID:AB_669740 KAP1 (rabbit) Bethyl Laboratories Cat# A300-274A; RRID:AB_185559 Alexa Fluor 568 goat anti-rabbit ThermoFisher Cat# A11036; RRID:AB_10563566 Alexa Fluor 594 goat anti-rabbit ThermoFisher Cat # A11012; RRID:AB_2534079 Alexa Fluor 488 goat anti-mouse ThermoFisher Cat# A11029; RRID:AB_2534088 Alexa Fluor 488 goat anti-mouse ThermoFisher Cat# A10667; RRID:AB_2534057 anti-mouse HRP-conjugated secondary antibody DAKO Cat# P0447; RRID:AB_2617137 anti-rabbit HRP-conjugated secondary antibody DAKO Cat# P0448; RRID:AB_2617138 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Sigma-Aldrich Cat# D5796 Tet system approved fetal bovine serum Takara Bio Cat# 631106 Doxycycline Sigma-Aldrich Cat# D9891 Tetracycline Sigma-Aldrich Cat# T7660 Thymidine Sigma-Aldrich Cat# T1895 RO-3306 Sigma-Aldrich Cat# SML0569 Hydroxyurea Sigma-Aldrich Cat# H8627 Nocodazole Sigma-Aldrich Cat# M1404 EdU ThermoFisher Cat# C10340 Aphidicolin Sigma-Aldrich Cat# A0781 DRB Sigma-Aldrich Cat# D1916 EU ThermoFisher Cat# E10345 INTERFERin (transfection reagent) Polyplus Cat# 409-01 Dharmafect1 (transfection reagent) Horizon Discovery Cat# T-2001-03 Azide-PEG(3+3)-S-S-biotin Jena Biosciences Cat# CLK-A2112-10 Dynabeads MyOne streptavidin C1 ThermoFisher Cat# 65001 b-mercaptoethanol Sigma-Aldrich Cat# M6250 TRIzol reagent ThermoFisher Cat# 15596026 HiMark Pre-stained Protein Standard ThermoFisher Cat# LC5699 (Continued on next page) e1 Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 .. REAGENT or RESOURCE SOURCE IDENTIFIER ProLong Gold Antifade Mountant ThermoFisher Cat# P36930 DAPI ThermoFisher Cat# D1306 Mowiol 4-88 Sigma-Aldrich Cat# 475904 Propidium iodide solution Sigma-Aldrich Cat# P4864 PureLink RNase A ThermoFisher Cat# 12091021 Critical commercial assays Click-iT EdU Alexa Fluor 647 Cell Proliferation Kit for Imaging ThermoFisher Cat# C10340 Click-iT Nascent RNA Capture Kit ThermoFisher Cat# C10365 Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit ThermoFisher Cat# C10424 TruSeq ChIP Library Preparation Kit Illumina Cat# IP-202-1012 TruSeq Stranded Total RNA with RiboZero Gold Illumina Cat# RS-122-2301 Deposited data DRIP-seq data (HeLa cells) Hamperl et al., 2017 GEO: GSM2452072 qDRIP-seq data (HeLa cells) Crossley et al., 2020 GEO: GSE134084 OK-seq data (RFD in HeLa cells) Petryk et al., 2016 https://github.com/CL-CHEN-Lab/OK- Seq/tree/master/published_results/HeLa EdU-seq BRCA2-deficient H1299 Dagg et al., 2021 GEO: GSM4650337 MiDAS-seq data This paper GEO: GSE196163 EU-seq data This paper GEO: GSE196163 Experimental models: Cell lines H1299 +shBRCA2DOX cells (male origin) Zimmer et al., 2016 N/A HeLa cells (female origin) ATCC Cat# CCL-2 HeLa +RNaseH1-FLAGDOX (female origin) Sollier et al., 2014 N/A U2OS CyclinE (tet OFF) RAD52+/+ (female origin) Sotiriou et al., 2016 N/A U2OS CyclinE (tet OFF) RAD52-/- (female origin) Sotiriou et al., 2016 N/A U2OS T-REx GFP-RnaseH1D210N (female origin) Teloni et al., 2019 N/A Oligonucleotides siGENOME BRCA2 siRNA (SMARTpool) Horizon Discovery Cat# M-003462-01 ON-TARGETplus RAD52 siRNA (SMARTpool) Horizon Discovery Cat# L-011760-00-0005 BRCA2 siRNA Qiagen Cat# SI02653434 AllStar negative control Qiagen Cat# 1027281 TP53 siRNA Dharmacon Cat# GEHCU-000146 ON-TARGETplus non-targeting control pool Horizon Discovery Cat# D-001810-10-05 Software and algorithms GraphPad Prism GraphPad https://www.graphpad.com/ Olympus ScanR Image Analysis Software 3.0.0 Olympus https://www.olympus-lifescience.com/en/ microscopes/inverted/scanr/ TIBCO Spotfire TIBCO Software https://www.tibco.com/products/tibco- spotfire Fiji Schindelin et al., 2012 https://fiji.sc/ Bedtools Quinlan and Hall, 2010 https://bedtools.readthedocs.io/en/latest/ (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 e2
Transfection:Article Title: Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RAD51 primary antibody (rabbit) BioAcademia Cat# 70-002 53BP1 primary antibody (rabbit) Novus Biologicals Cat# NB100-304; RRID:AB_10003037 53BP1 primary antibody (mouse) Thanos Halazonetis (Schultz et al., 2000) N/A H2AX Phospho S139 primary antibody (rabbit) Cell Signaling Cat# 9718; RRID:AB_2118009 Histone H3 Phospho S10 primary antibody (mouse) Cell Signaling Cat# 9701; RRID:AB_331535 BRCA2 primary antibody (mouse) Calbiochem Cat# OP95; RRID:AB_2067762 SMC1 primary antibody (rabbit) Bethyl Laboratories Cat# A300-055A; RRID:AB_2192467 GAPDH primary antibody (mouse) Novus Biologicals Cat# NB600-502; RRID:AB_10077682 FLAG primary antibody (mouse) Agilent Technologies (Stratagene) Cat# 200470-21 KAP1 Phospho S824 primary antibody (rabbit) Bethyl Laboratories Cat# A300-767A; RRID:AB_669740 KAP1 (rabbit) Bethyl Laboratories Cat# A300-274A; RRID:AB_185559 Alexa Fluor 568 goat anti-rabbit ThermoFisher Cat# A11036; RRID:AB_10563566 Alexa Fluor 594 goat anti-rabbit ThermoFisher Cat # A11012; RRID:AB_2534079 Alexa Fluor 488 goat anti-mouse ThermoFisher Cat# A11029; RRID:AB_2534088 Alexa Fluor 488 goat anti-mouse ThermoFisher Cat# A10667; RRID:AB_2534057 anti-mouse HRP-conjugated secondary antibody DAKO Cat# P0447; RRID:AB_2617137 anti-rabbit HRP-conjugated secondary antibody DAKO Cat# P0448; RRID:AB_2617138 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Sigma-Aldrich Cat# D5796 Tet system approved fetal bovine serum Takara Bio Cat# 631106 Doxycycline Sigma-Aldrich Cat# D9891 Tetracycline Sigma-Aldrich Cat# T7660 Thymidine Sigma-Aldrich Cat# T1895 RO-3306 Sigma-Aldrich Cat# SML0569 Hydroxyurea Sigma-Aldrich Cat# H8627 Nocodazole Sigma-Aldrich Cat# M1404 EdU ThermoFisher Cat# C10340 Aphidicolin Sigma-Aldrich Cat# A0781 DRB Sigma-Aldrich Cat# D1916 EU ThermoFisher Cat# E10345 INTERFERin (transfection reagent) Polyplus Cat# 409-01 Dharmafect1 (transfection reagent) Horizon Discovery Cat# T-2001-03 Azide-PEG(3+3)-S-S-biotin Jena Biosciences Cat# CLK-A2112-10 Dynabeads MyOne streptavidin C1 ThermoFisher Cat# 65001 b-mercaptoethanol Sigma-Aldrich Cat# M6250 TRIzol reagent ThermoFisher Cat# 15596026 HiMark Pre-stained Protein Standard ThermoFisher Cat# LC5699 (Continued on next page) e1 Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 .. REAGENT or RESOURCE SOURCE IDENTIFIER ProLong Gold Antifade Mountant ThermoFisher Cat# P36930 DAPI ThermoFisher Cat# D1306 Mowiol 4-88 Sigma-Aldrich Cat# 475904 Propidium iodide solution Sigma-Aldrich Cat# P4864 PureLink RNase A ThermoFisher Cat# 12091021 Critical commercial assays Click-iT EdU Alexa Fluor 647 Cell Proliferation Kit for Imaging ThermoFisher Cat# C10340 Click-iT Nascent RNA Capture Kit ThermoFisher Cat# C10365 Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit ThermoFisher Cat# C10424 TruSeq ChIP Library Preparation Kit Illumina Cat# IP-202-1012 TruSeq Stranded Total RNA with RiboZero Gold Illumina Cat# RS-122-2301 Deposited data DRIP-seq data (HeLa cells) Hamperl et al., 2017 GEO: GSM2452072 qDRIP-seq data (HeLa cells) Crossley et al., 2020 GEO: GSE134084 OK-seq data (RFD in HeLa cells) Petryk et al., 2016 https://github.com/CL-CHEN-Lab/OK- Seq/tree/master/published_results/HeLa EdU-seq BRCA2-deficient H1299 Dagg et al., 2021 GEO: GSM4650337 MiDAS-seq data This paper GEO: GSE196163 EU-seq data This paper GEO: GSE196163 Experimental models: Cell lines H1299 +shBRCA2DOX cells (male origin) Zimmer et al., 2016 N/A HeLa cells (female origin) ATCC Cat# CCL-2 HeLa +RNaseH1-FLAGDOX (female origin) Sollier et al., 2014 N/A U2OS CyclinE (tet OFF) RAD52+/+ (female origin) Sotiriou et al., 2016 N/A U2OS CyclinE (tet OFF) RAD52-/- (female origin) Sotiriou et al., 2016 N/A U2OS T-REx GFP-RnaseH1D210N (female origin) Teloni et al., 2019 N/A Oligonucleotides siGENOME BRCA2 siRNA (SMARTpool) Horizon Discovery Cat# M-003462-01 ON-TARGETplus RAD52 siRNA (SMARTpool) Horizon Discovery Cat# L-011760-00-0005 BRCA2 siRNA Qiagen Cat# SI02653434 AllStar negative control Qiagen Cat# 1027281 TP53 siRNA Dharmacon Cat# GEHCU-000146 ON-TARGETplus non-targeting control pool Horizon Discovery Cat# D-001810-10-05 Software and algorithms GraphPad Prism GraphPad https://www.graphpad.com/ Olympus ScanR Image Analysis Software 3.0.0 Olympus https://www.olympus-lifescience.com/en/ microscopes/inverted/scanr/ TIBCO Spotfire TIBCO Software https://www.tibco.com/products/tibco- spotfire Fiji Schindelin et al., 2012 https://fiji.sc/ Bedtools Quinlan and Hall, 2010 https://bedtools.readthedocs.io/en/latest/ (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 e2
Western Blot:
Chromatin Immunoprecipitation:Article Title: CTCF controls three-dimensional enhancer network underlying the inflammatory response of bone marrow-derived dendritic cells
Article Snippet: Validated ChIP in human cell lines on the manufacturer's website anti-H3K4me3 (ChIP-seq): Abcam ab8580, lot GR288375-1. .. Validated ChIP in human cell lines on the manufacturer's website anti-SMC1 (ChIP-seq): Bethyl lab A300-055A, lot 6. ..
other:
Biomarker Discovery:
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Bethyl is a verified supplier 