GSM5183572: DNMT3A ChIP-seq #1 on H1 QSER1/TET1 DKO hESCs; Homo sapiens; ChIP-Seq
ChIP-seq was performed as follows. Cells were cross-linked with 1% formaldehyde (Sigma, F1635) at 37 °C for 15 minutes and quenched with 0.125 M glycine for 5 minutes at room temperature. Fixed cells were scraped off the plates in cold PBS buffer and washed twice in cold PBS buffer. Cell pellets were obtained by centrifugation at 3,000 RPM for 5 minutes at 4 °C and frozen in liquid nitrogen immediately before transferring to the −80 °C freezer. ~25 million cells were used for one ChIP reaction. Cell pellets were thawed on ice, resuspended in 1 ml SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8) containing proteinase and phosphatase inhibitor for one reaction in an Eppendorf tube and incubated on ice for 10 minutes. Sonication was performed on a Branson Sonifier 250 with a 20% amplitude setting for 5.5 minutes (10 seconds on/off pulsing). Sonication products were spun down at 14,000 RPM at 4 °C for 10 minutes and 1 ml supernatant containing chromatin and DNA were transferred to a falcon tube containing 9 ml ChIP dilution buffer (0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl) with proteinase and phosphatase inhibitor. 50 μl of Dynabeads (Life Technologies, 10009D) were added to samples and incubated at 4 °C with rotation for 1 hour. After pre-clearing, Dynabeads beads were removed and 200 μl of sample were collected as 2% input separately. 5 μg antibodies were added to the pre-cleared samples for overnight incubation at 4 °C with rotation. 200 μl Dynabeads were added into one ChIP reaction and incubated for 4–6 hours at 4 °C with rotation. Dynabeads were collected by centrifugation with 3,000 RPM at 4 °C for 5 minutes and washed in 1 ml low salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl) for 5 minutes at 4 °C with rotations. Then beads were washed in 1 ml high salt buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 500 mM NaCl) twice and TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA) twice for 5 minutes at 4 °C with rotation. After the last wash, beads were resuspended in 250 μl elution buffer (1% SDS, 0.1 M NaHCO3) and incubated in a thermomixer: 850 RPM for 15 minutes at 60 °C. Supernatant were collected and added with 5 M NaCl for overnight decrosslinking at 65 °C. 10 μl 0.5 M EDTA, 20 μl 1 M Tris-HCl pH6.5 and 1 μl proteinase K (20 mg/ml) were added to decrosslinked product and incubated for 1 hour at 45 °C. DNA was isolated by using QIAquick PCR purification kit (Qiagen, 28104). Libraries were prepared using the NEBNext® Ultra II DNA Library Prep Kit (NEB, E7103S) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1; NEB, E7335S). Samples were pooled and submitted to MSKCC Integrated Genomics Operation core for quality control and sequencing as follows. After PicoGreen quantification and quality control by Agilent BioAnalyzer, pooled libraries were run over one lane of a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina). The loading concentration was 2nM and a 5% spike-in of PhiX was added to the run to increase diversity and for quality control purposes. The lane yielded ~269M reads.
GEO Accession
GSM5183572