Polymerase Chain Reaction:Article Title: Robust Bisulfite‐Free Single‐Molecule Real‐Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme
Article Snippet: The finished ligation reaction was directly subjected to bisulfite treatment according to the manufacturer’s protocol using the EZ DNA Methylation-GoldTM Kit (Zymo Research, cat. no. D5005). .. The DNA was eluted on the bench in 15 μl prewarmed (55°C) M-Elution Buffer (Zymo Research) for 5 min. For Polymerase Chain Reaction (PCR), 10 μl bisulfite-treated eluted DNA, 12.5 μl Q5U Master Mix (NEB, cat. no. M0597S), 1.25 μl 10 μM Truseq Forward Primer (Microsynth) and 1.25 μl 10 μM Truseq Reverse Primer (Microsynth) were mixed by pipetting and incubated at the following condition- Temperature Time Cycle 98oC 30 sec 98oC 15 sec 14 58oC 40 sec 72oC 30 sec 72oC 5 min 16oC ∞ Subsequently, the PCR product was purified with 0.9x AMPure XP beads (Beckman Coulter) according to the manufacturer’s protocol and the DNA was eluted at 37 °C in 15 μl prewarmed (at 55°C) Elution buffer (PacBio) for 5 min. .. The concentration of the final library was determined using the Qubit dsDNAHS-Assay-Kit (ThermoFisher, cat. no. Q32851) and the DNA length and quality of the final library were determined using the High Sensitivity D1000 ScreenTape assay (Agilent).
Article Title: Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain
Article Snippet: snm3C/snmC-seq3 Beckman i7 Protocol – Ecker Lab MATERIALS- General reagents • FACS sorted single-nuclei into 384-well PCR plate • HyCloneTM HyPureTM Molecular Biology Grade (MB) Water (GE Life Sci. .. SH30538.08) • 200-Proof (100%) Ethanol (Koptec V1001) Collection of single nuclei by Fluorescence-activated Cell Sorting (FACS) • M-Digestion Buffer (Zymo D5021-9) • Proteinase K w/Storage Buffer (Zymo D3001-2-20) • CpG Methyltransferase (M.SssI) (NEB M0226) • Unmethylated Lambda DNA (Promega D1521) Bisulfite Conversion and Cleanup • CT Conversion Reagent (Zymo D5003-1) • M-Solubilization Buffer (Zymo D5021-7) • M-Dilution Buffer-Gold (Zymo D5006-2) • M-Reaction Buffer (Zymo D5021-8) • MagBead J (Zymo custom product D4100-3-100) • M-Binding Buffer (Zymo D5021-7) • M-Wash Buffer (Zymo D5040-4) • M-Desulphonation Buffer (Zymo D5040-5) • M-Elution Buffer (Zymo D5007-6) Random primers • Random Indexing Primer (IDT custom DNA oligo, standard desalted) Sera-Mag Solid Phase Reversible Immobilization (SPRI) beads • Sera-Mag SpeedBeads Magnetic Carboxylate Modified (GE Healthcare 5152105050250) • Poly(ethylene glycol) PEG 8000 (Sigma cat no. 89510-250G-F) • TE buffer pH=8.0 (Ambion AM9858) • 5M NaCl • 1M Tris-HCl pH=8.0 • 0.5M EDTA pH=8.0 • AMPure XP beads (Beckman Coulter cat no. A63881) • 100 bp DNA ladder (New England Biolabs cat no. N3231L) snmC-seq3 Library Preparation • Blue Buffer (10x) (Enzymatics P7010-HC-L) • Klenow Exo- (50U/μL) (Enzymatics P7010-HC-L) • Deoxynucleotide Solution Mix (10mM each dNTP) (NEB N0447L) • Exonuclease I (20U/μL) (Enzymatics X8010L) • Shrimp Alkaline Phosphatase (rSAP) (1U/μL) (NEB M0371L) • Sera-Mag SPRI beads • M-Elution Buffer (Zymo 5007-6) • EB buffer (Qiagen 19086) • Accel-NGS® AdaptaseTM Module (Swift Bio 330384) • P5 Indexing Primer (IDT custom DNA oligo, standard desalted) • P7 Indexing Primer (IDT custom DNA oligo, standard desalted) • KAPA HiFi HS RM (Kapa KK2602) • Qubit dsDNA BR Assay Kit (Thermo Fisher Q32850) Equipment • 384-Well Hardshell PCR Plate Clear, 20 Pcs/pk (Thermo Fisher 4483285) • 96-Well Hardshell PCR Plate GPLE, 20 Pcs/pk (Thermo Fisher 4483348) • MicroAmpTM Splash-Free 96-Well Base (Thermo Fisher 4312063) • Plateone® Deep 384-Well 200 μL Polypropylene Plate (USA-SCI. .. 1884-2410) • Reservoir Single Well 384 Bottom Low Profile, Clear (Axygen RES-SW384-LP) • Reservoir Single Well 96 Bottom High Profile, Clear (Axygen RES-SW96-HP) • Reservoir Single Well 96 Bottom Low Profile, Clear (Axygen RES-SW96-LP) • 15mL Centrifuge Tubes (Olympus 28-103) • 50mL Centrifuge Tubes (Olympus 28-106) • 1.7mL Microtube, Clear (Olympus 24-282LR) • Microamp Clear Adhesive Film, 100pc (Thermo Fisher 4306311) • Speedball Deluxe Soft Rubber Brayer, 4 inches (Statesville N.C.) • 37°C Incubator • 384-well and 96-well Compatible Thermocycler • DynaMagTM-96 Side Magnet (Thermo Fisher 12331D) • DynaMagTM-2 Magnet (Thermo Fisher 12321D) • 384-well Magnet Plate (Alpaqua A001222) • Allegra 25R Centrifuge with S5700 2x96 Swinging Bucket Rotor (Beckman Coulter 368954) • Axygen® 384-well tips, 45μL, Clear, Filtered, Sterile, Fine Point, SLAS Rack (Corning FXF-384- 45FP-R-S) • Axygen® 96-well tips, 200μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-200-R-S) • Axygen® 96-well tips, 30μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-50-R-S) • Beckman Coulter Biomek i7 dual-Multichannel with MC384 and MC96 Pipetting Heads REAGENT SETUP- Methylated λ DNA Dilute supplied SAM to 1600μM with Nuclease-free Water.
Incubation:Article Title: Robust Bisulfite‐Free Single‐Molecule Real‐Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme
Article Snippet: The finished ligation reaction was directly subjected to bisulfite treatment according to the manufacturer’s protocol using the EZ DNA Methylation-GoldTM Kit (Zymo Research, cat. no. D5005). .. The DNA was eluted on the bench in 15 μl prewarmed (55°C) M-Elution Buffer (Zymo Research) for 5 min. For Polymerase Chain Reaction (PCR), 10 μl bisulfite-treated eluted DNA, 12.5 μl Q5U Master Mix (NEB, cat. no. M0597S), 1.25 μl 10 μM Truseq Forward Primer (Microsynth) and 1.25 μl 10 μM Truseq Reverse Primer (Microsynth) were mixed by pipetting and incubated at the following condition- Temperature Time Cycle 98oC 30 sec 98oC 15 sec 14 58oC 40 sec 72oC 30 sec 72oC 5 min 16oC ∞ Subsequently, the PCR product was purified with 0.9x AMPure XP beads (Beckman Coulter) according to the manufacturer’s protocol and the DNA was eluted at 37 °C in 15 μl prewarmed (at 55°C) Elution buffer (PacBio) for 5 min. .. The concentration of the final library was determined using the Qubit dsDNAHS-Assay-Kit (ThermoFisher, cat. no. Q32851) and the DNA length and quality of the final library were determined using the High Sensitivity D1000 ScreenTape assay (Agilent).
Purification:Article Title: Robust Bisulfite‐Free Single‐Molecule Real‐Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme
Article Snippet: The finished ligation reaction was directly subjected to bisulfite treatment according to the manufacturer’s protocol using the EZ DNA Methylation-GoldTM Kit (Zymo Research, cat. no. D5005). .. The DNA was eluted on the bench in 15 μl prewarmed (55°C) M-Elution Buffer (Zymo Research) for 5 min. For Polymerase Chain Reaction (PCR), 10 μl bisulfite-treated eluted DNA, 12.5 μl Q5U Master Mix (NEB, cat. no. M0597S), 1.25 μl 10 μM Truseq Forward Primer (Microsynth) and 1.25 μl 10 μM Truseq Reverse Primer (Microsynth) were mixed by pipetting and incubated at the following condition- Temperature Time Cycle 98oC 30 sec 98oC 15 sec 14 58oC 40 sec 72oC 30 sec 72oC 5 min 16oC ∞ Subsequently, the PCR product was purified with 0.9x AMPure XP beads (Beckman Coulter) according to the manufacturer’s protocol and the DNA was eluted at 37 °C in 15 μl prewarmed (at 55°C) Elution buffer (PacBio) for 5 min. .. The concentration of the final library was determined using the Qubit dsDNAHS-Assay-Kit (ThermoFisher, cat. no. Q32851) and the DNA length and quality of the final library were determined using the High Sensitivity D1000 ScreenTape assay (Agilent).
Fluorescence:Article Title: Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain
Article Snippet: snm3C/snmC-seq3 Beckman i7 Protocol – Ecker Lab MATERIALS- General reagents • FACS sorted single-nuclei into 384-well PCR plate • HyCloneTM HyPureTM Molecular Biology Grade (MB) Water (GE Life Sci. .. SH30538.08) • 200-Proof (100%) Ethanol (Koptec V1001) Collection of single nuclei by Fluorescence-activated Cell Sorting (FACS) • M-Digestion Buffer (Zymo D5021-9) • Proteinase K w/Storage Buffer (Zymo D3001-2-20) • CpG Methyltransferase (M.SssI) (NEB M0226) • Unmethylated Lambda DNA (Promega D1521) Bisulfite Conversion and Cleanup • CT Conversion Reagent (Zymo D5003-1) • M-Solubilization Buffer (Zymo D5021-7) • M-Dilution Buffer-Gold (Zymo D5006-2) • M-Reaction Buffer (Zymo D5021-8) • MagBead J (Zymo custom product D4100-3-100) • M-Binding Buffer (Zymo D5021-7) • M-Wash Buffer (Zymo D5040-4) • M-Desulphonation Buffer (Zymo D5040-5) • M-Elution Buffer (Zymo D5007-6) Random primers • Random Indexing Primer (IDT custom DNA oligo, standard desalted) Sera-Mag Solid Phase Reversible Immobilization (SPRI) beads • Sera-Mag SpeedBeads Magnetic Carboxylate Modified (GE Healthcare 5152105050250) • Poly(ethylene glycol) PEG 8000 (Sigma cat no. 89510-250G-F) • TE buffer pH=8.0 (Ambion AM9858) • 5M NaCl • 1M Tris-HCl pH=8.0 • 0.5M EDTA pH=8.0 • AMPure XP beads (Beckman Coulter cat no. A63881) • 100 bp DNA ladder (New England Biolabs cat no. N3231L) snmC-seq3 Library Preparation • Blue Buffer (10x) (Enzymatics P7010-HC-L) • Klenow Exo- (50U/μL) (Enzymatics P7010-HC-L) • Deoxynucleotide Solution Mix (10mM each dNTP) (NEB N0447L) • Exonuclease I (20U/μL) (Enzymatics X8010L) • Shrimp Alkaline Phosphatase (rSAP) (1U/μL) (NEB M0371L) • Sera-Mag SPRI beads • M-Elution Buffer (Zymo 5007-6) • EB buffer (Qiagen 19086) • Accel-NGS® AdaptaseTM Module (Swift Bio 330384) • P5 Indexing Primer (IDT custom DNA oligo, standard desalted) • P7 Indexing Primer (IDT custom DNA oligo, standard desalted) • KAPA HiFi HS RM (Kapa KK2602) • Qubit dsDNA BR Assay Kit (Thermo Fisher Q32850) Equipment • 384-Well Hardshell PCR Plate Clear, 20 Pcs/pk (Thermo Fisher 4483285) • 96-Well Hardshell PCR Plate GPLE, 20 Pcs/pk (Thermo Fisher 4483348) • MicroAmpTM Splash-Free 96-Well Base (Thermo Fisher 4312063) • Plateone® Deep 384-Well 200 μL Polypropylene Plate (USA-SCI. .. 1884-2410) • Reservoir Single Well 384 Bottom Low Profile, Clear (Axygen RES-SW384-LP) • Reservoir Single Well 96 Bottom High Profile, Clear (Axygen RES-SW96-HP) • Reservoir Single Well 96 Bottom Low Profile, Clear (Axygen RES-SW96-LP) • 15mL Centrifuge Tubes (Olympus 28-103) • 50mL Centrifuge Tubes (Olympus 28-106) • 1.7mL Microtube, Clear (Olympus 24-282LR) • Microamp Clear Adhesive Film, 100pc (Thermo Fisher 4306311) • Speedball Deluxe Soft Rubber Brayer, 4 inches (Statesville N.C.) • 37°C Incubator • 384-well and 96-well Compatible Thermocycler • DynaMagTM-96 Side Magnet (Thermo Fisher 12331D) • DynaMagTM-2 Magnet (Thermo Fisher 12321D) • 384-well Magnet Plate (Alpaqua A001222) • Allegra 25R Centrifuge with S5700 2x96 Swinging Bucket Rotor (Beckman Coulter 368954) • Axygen® 384-well tips, 45μL, Clear, Filtered, Sterile, Fine Point, SLAS Rack (Corning FXF-384- 45FP-R-S) • Axygen® 96-well tips, 200μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-200-R-S) • Axygen® 96-well tips, 30μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-50-R-S) • Beckman Coulter Biomek i7 dual-Multichannel with MC384 and MC96 Pipetting Heads REAGENT SETUP- Methylated λ DNA Dilute supplied SAM to 1600μM with Nuclease-free Water.
FACS:Article Title: Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain
Article Snippet: snm3C/snmC-seq3 Beckman i7 Protocol – Ecker Lab MATERIALS- General reagents • FACS sorted single-nuclei into 384-well PCR plate • HyCloneTM HyPureTM Molecular Biology Grade (MB) Water (GE Life Sci. .. SH30538.08) • 200-Proof (100%) Ethanol (Koptec V1001) Collection of single nuclei by Fluorescence-activated Cell Sorting (FACS) • M-Digestion Buffer (Zymo D5021-9) • Proteinase K w/Storage Buffer (Zymo D3001-2-20) • CpG Methyltransferase (M.SssI) (NEB M0226) • Unmethylated Lambda DNA (Promega D1521) Bisulfite Conversion and Cleanup • CT Conversion Reagent (Zymo D5003-1) • M-Solubilization Buffer (Zymo D5021-7) • M-Dilution Buffer-Gold (Zymo D5006-2) • M-Reaction Buffer (Zymo D5021-8) • MagBead J (Zymo custom product D4100-3-100) • M-Binding Buffer (Zymo D5021-7) • M-Wash Buffer (Zymo D5040-4) • M-Desulphonation Buffer (Zymo D5040-5) • M-Elution Buffer (Zymo D5007-6) Random primers • Random Indexing Primer (IDT custom DNA oligo, standard desalted) Sera-Mag Solid Phase Reversible Immobilization (SPRI) beads • Sera-Mag SpeedBeads Magnetic Carboxylate Modified (GE Healthcare 5152105050250) • Poly(ethylene glycol) PEG 8000 (Sigma cat no. 89510-250G-F) • TE buffer pH=8.0 (Ambion AM9858) • 5M NaCl • 1M Tris-HCl pH=8.0 • 0.5M EDTA pH=8.0 • AMPure XP beads (Beckman Coulter cat no. A63881) • 100 bp DNA ladder (New England Biolabs cat no. N3231L) snmC-seq3 Library Preparation • Blue Buffer (10x) (Enzymatics P7010-HC-L) • Klenow Exo- (50U/μL) (Enzymatics P7010-HC-L) • Deoxynucleotide Solution Mix (10mM each dNTP) (NEB N0447L) • Exonuclease I (20U/μL) (Enzymatics X8010L) • Shrimp Alkaline Phosphatase (rSAP) (1U/μL) (NEB M0371L) • Sera-Mag SPRI beads • M-Elution Buffer (Zymo 5007-6) • EB buffer (Qiagen 19086) • Accel-NGS® AdaptaseTM Module (Swift Bio 330384) • P5 Indexing Primer (IDT custom DNA oligo, standard desalted) • P7 Indexing Primer (IDT custom DNA oligo, standard desalted) • KAPA HiFi HS RM (Kapa KK2602) • Qubit dsDNA BR Assay Kit (Thermo Fisher Q32850) Equipment • 384-Well Hardshell PCR Plate Clear, 20 Pcs/pk (Thermo Fisher 4483285) • 96-Well Hardshell PCR Plate GPLE, 20 Pcs/pk (Thermo Fisher 4483348) • MicroAmpTM Splash-Free 96-Well Base (Thermo Fisher 4312063) • Plateone® Deep 384-Well 200 μL Polypropylene Plate (USA-SCI. .. 1884-2410) • Reservoir Single Well 384 Bottom Low Profile, Clear (Axygen RES-SW384-LP) • Reservoir Single Well 96 Bottom High Profile, Clear (Axygen RES-SW96-HP) • Reservoir Single Well 96 Bottom Low Profile, Clear (Axygen RES-SW96-LP) • 15mL Centrifuge Tubes (Olympus 28-103) • 50mL Centrifuge Tubes (Olympus 28-106) • 1.7mL Microtube, Clear (Olympus 24-282LR) • Microamp Clear Adhesive Film, 100pc (Thermo Fisher 4306311) • Speedball Deluxe Soft Rubber Brayer, 4 inches (Statesville N.C.) • 37°C Incubator • 384-well and 96-well Compatible Thermocycler • DynaMagTM-96 Side Magnet (Thermo Fisher 12331D) • DynaMagTM-2 Magnet (Thermo Fisher 12321D) • 384-well Magnet Plate (Alpaqua A001222) • Allegra 25R Centrifuge with S5700 2x96 Swinging Bucket Rotor (Beckman Coulter 368954) • Axygen® 384-well tips, 45μL, Clear, Filtered, Sterile, Fine Point, SLAS Rack (Corning FXF-384- 45FP-R-S) • Axygen® 96-well tips, 200μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-200-R-S) • Axygen® 96-well tips, 30μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-50-R-S) • Beckman Coulter Biomek i7 dual-Multichannel with MC384 and MC96 Pipetting Heads REAGENT SETUP- Methylated λ DNA Dilute supplied SAM to 1600μM with Nuclease-free Water.
Lambda DNA Preparation:Article Title: Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain
Article Snippet: snm3C/snmC-seq3 Beckman i7 Protocol – Ecker Lab MATERIALS- General reagents • FACS sorted single-nuclei into 384-well PCR plate • HyCloneTM HyPureTM Molecular Biology Grade (MB) Water (GE Life Sci. .. SH30538.08) • 200-Proof (100%) Ethanol (Koptec V1001) Collection of single nuclei by Fluorescence-activated Cell Sorting (FACS) • M-Digestion Buffer (Zymo D5021-9) • Proteinase K w/Storage Buffer (Zymo D3001-2-20) • CpG Methyltransferase (M.SssI) (NEB M0226) • Unmethylated Lambda DNA (Promega D1521) Bisulfite Conversion and Cleanup • CT Conversion Reagent (Zymo D5003-1) • M-Solubilization Buffer (Zymo D5021-7) • M-Dilution Buffer-Gold (Zymo D5006-2) • M-Reaction Buffer (Zymo D5021-8) • MagBead J (Zymo custom product D4100-3-100) • M-Binding Buffer (Zymo D5021-7) • M-Wash Buffer (Zymo D5040-4) • M-Desulphonation Buffer (Zymo D5040-5) • M-Elution Buffer (Zymo D5007-6) Random primers • Random Indexing Primer (IDT custom DNA oligo, standard desalted) Sera-Mag Solid Phase Reversible Immobilization (SPRI) beads • Sera-Mag SpeedBeads Magnetic Carboxylate Modified (GE Healthcare 5152105050250) • Poly(ethylene glycol) PEG 8000 (Sigma cat no. 89510-250G-F) • TE buffer pH=8.0 (Ambion AM9858) • 5M NaCl • 1M Tris-HCl pH=8.0 • 0.5M EDTA pH=8.0 • AMPure XP beads (Beckman Coulter cat no. A63881) • 100 bp DNA ladder (New England Biolabs cat no. N3231L) snmC-seq3 Library Preparation • Blue Buffer (10x) (Enzymatics P7010-HC-L) • Klenow Exo- (50U/μL) (Enzymatics P7010-HC-L) • Deoxynucleotide Solution Mix (10mM each dNTP) (NEB N0447L) • Exonuclease I (20U/μL) (Enzymatics X8010L) • Shrimp Alkaline Phosphatase (rSAP) (1U/μL) (NEB M0371L) • Sera-Mag SPRI beads • M-Elution Buffer (Zymo 5007-6) • EB buffer (Qiagen 19086) • Accel-NGS® AdaptaseTM Module (Swift Bio 330384) • P5 Indexing Primer (IDT custom DNA oligo, standard desalted) • P7 Indexing Primer (IDT custom DNA oligo, standard desalted) • KAPA HiFi HS RM (Kapa KK2602) • Qubit dsDNA BR Assay Kit (Thermo Fisher Q32850) Equipment • 384-Well Hardshell PCR Plate Clear, 20 Pcs/pk (Thermo Fisher 4483285) • 96-Well Hardshell PCR Plate GPLE, 20 Pcs/pk (Thermo Fisher 4483348) • MicroAmpTM Splash-Free 96-Well Base (Thermo Fisher 4312063) • Plateone® Deep 384-Well 200 μL Polypropylene Plate (USA-SCI. .. 1884-2410) • Reservoir Single Well 384 Bottom Low Profile, Clear (Axygen RES-SW384-LP) • Reservoir Single Well 96 Bottom High Profile, Clear (Axygen RES-SW96-HP) • Reservoir Single Well 96 Bottom Low Profile, Clear (Axygen RES-SW96-LP) • 15mL Centrifuge Tubes (Olympus 28-103) • 50mL Centrifuge Tubes (Olympus 28-106) • 1.7mL Microtube, Clear (Olympus 24-282LR) • Microamp Clear Adhesive Film, 100pc (Thermo Fisher 4306311) • Speedball Deluxe Soft Rubber Brayer, 4 inches (Statesville N.C.) • 37°C Incubator • 384-well and 96-well Compatible Thermocycler • DynaMagTM-96 Side Magnet (Thermo Fisher 12331D) • DynaMagTM-2 Magnet (Thermo Fisher 12321D) • 384-well Magnet Plate (Alpaqua A001222) • Allegra 25R Centrifuge with S5700 2x96 Swinging Bucket Rotor (Beckman Coulter 368954) • Axygen® 384-well tips, 45μL, Clear, Filtered, Sterile, Fine Point, SLAS Rack (Corning FXF-384- 45FP-R-S) • Axygen® 96-well tips, 200μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-200-R-S) • Axygen® 96-well tips, 30μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-50-R-S) • Beckman Coulter Biomek i7 dual-Multichannel with MC384 and MC96 Pipetting Heads REAGENT SETUP- Methylated λ DNA Dilute supplied SAM to 1600μM with Nuclease-free Water.
Modification:Article Title: Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain
Article Snippet: snm3C/snmC-seq3 Beckman i7 Protocol – Ecker Lab MATERIALS- General reagents • FACS sorted single-nuclei into 384-well PCR plate • HyCloneTM HyPureTM Molecular Biology Grade (MB) Water (GE Life Sci. .. SH30538.08) • 200-Proof (100%) Ethanol (Koptec V1001) Collection of single nuclei by Fluorescence-activated Cell Sorting (FACS) • M-Digestion Buffer (Zymo D5021-9) • Proteinase K w/Storage Buffer (Zymo D3001-2-20) • CpG Methyltransferase (M.SssI) (NEB M0226) • Unmethylated Lambda DNA (Promega D1521) Bisulfite Conversion and Cleanup • CT Conversion Reagent (Zymo D5003-1) • M-Solubilization Buffer (Zymo D5021-7) • M-Dilution Buffer-Gold (Zymo D5006-2) • M-Reaction Buffer (Zymo D5021-8) • MagBead J (Zymo custom product D4100-3-100) • M-Binding Buffer (Zymo D5021-7) • M-Wash Buffer (Zymo D5040-4) • M-Desulphonation Buffer (Zymo D5040-5) • M-Elution Buffer (Zymo D5007-6) Random primers • Random Indexing Primer (IDT custom DNA oligo, standard desalted) Sera-Mag Solid Phase Reversible Immobilization (SPRI) beads • Sera-Mag SpeedBeads Magnetic Carboxylate Modified (GE Healthcare 5152105050250) • Poly(ethylene glycol) PEG 8000 (Sigma cat no. 89510-250G-F) • TE buffer pH=8.0 (Ambion AM9858) • 5M NaCl • 1M Tris-HCl pH=8.0 • 0.5M EDTA pH=8.0 • AMPure XP beads (Beckman Coulter cat no. A63881) • 100 bp DNA ladder (New England Biolabs cat no. N3231L) snmC-seq3 Library Preparation • Blue Buffer (10x) (Enzymatics P7010-HC-L) • Klenow Exo- (50U/μL) (Enzymatics P7010-HC-L) • Deoxynucleotide Solution Mix (10mM each dNTP) (NEB N0447L) • Exonuclease I (20U/μL) (Enzymatics X8010L) • Shrimp Alkaline Phosphatase (rSAP) (1U/μL) (NEB M0371L) • Sera-Mag SPRI beads • M-Elution Buffer (Zymo 5007-6) • EB buffer (Qiagen 19086) • Accel-NGS® AdaptaseTM Module (Swift Bio 330384) • P5 Indexing Primer (IDT custom DNA oligo, standard desalted) • P7 Indexing Primer (IDT custom DNA oligo, standard desalted) • KAPA HiFi HS RM (Kapa KK2602) • Qubit dsDNA BR Assay Kit (Thermo Fisher Q32850) Equipment • 384-Well Hardshell PCR Plate Clear, 20 Pcs/pk (Thermo Fisher 4483285) • 96-Well Hardshell PCR Plate GPLE, 20 Pcs/pk (Thermo Fisher 4483348) • MicroAmpTM Splash-Free 96-Well Base (Thermo Fisher 4312063) • Plateone® Deep 384-Well 200 μL Polypropylene Plate (USA-SCI. .. 1884-2410) • Reservoir Single Well 384 Bottom Low Profile, Clear (Axygen RES-SW384-LP) • Reservoir Single Well 96 Bottom High Profile, Clear (Axygen RES-SW96-HP) • Reservoir Single Well 96 Bottom Low Profile, Clear (Axygen RES-SW96-LP) • 15mL Centrifuge Tubes (Olympus 28-103) • 50mL Centrifuge Tubes (Olympus 28-106) • 1.7mL Microtube, Clear (Olympus 24-282LR) • Microamp Clear Adhesive Film, 100pc (Thermo Fisher 4306311) • Speedball Deluxe Soft Rubber Brayer, 4 inches (Statesville N.C.) • 37°C Incubator • 384-well and 96-well Compatible Thermocycler • DynaMagTM-96 Side Magnet (Thermo Fisher 12331D) • DynaMagTM-2 Magnet (Thermo Fisher 12321D) • 384-well Magnet Plate (Alpaqua A001222) • Allegra 25R Centrifuge with S5700 2x96 Swinging Bucket Rotor (Beckman Coulter 368954) • Axygen® 384-well tips, 45μL, Clear, Filtered, Sterile, Fine Point, SLAS Rack (Corning FXF-384- 45FP-R-S) • Axygen® 96-well tips, 200μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-200-R-S) • Axygen® 96-well tips, 30μL, Clear, Filtered, Sterile, SLAS Rack (Corning FXF-50-R-S) • Beckman Coulter Biomek i7 dual-Multichannel with MC384 and MC96 Pipetting Heads REAGENT SETUP- Methylated λ DNA Dilute supplied SAM to 1600μM with Nuclease-free Water.
Methylation:
|
Zymo Research is a verified supplier 
Zymo Research manufactures this product 