GSM5219549: KI H3K4me3 0 rep 2; Homo sapiens; ChIP-Seq
Briefly 2.5x10^6 cells were crosslinked with 2mM DSG in PBS for 50 min with gentle agitation, washed with PBS and incubated in PBS-1% formaldehyde for 5 minutes. Cross-linking was stopped by the addition of 125 mM glycine (final concentration) and cells were washed twice with cold PBS. After harvesting cells by scraping (attached cells) and centrifugation (detached cells), the pellet was washed once with PBS plus 0.5% BSA and resuspended in TE-SDS 0.1% (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, SDS 0.1%) supplemented with complete protease inhibitor tablet (Roche) to a final concentration of 2.5x10^6 cells/ml. Samples were sonicated for 20 minutes with Covaris ME220 sonicator using the 1ml High Cell protocol (Peak power: 75, Duty % factor: 15, Cycles/Burst: 1000, Average power: 11.25) to produce chromatin fragments of 200-400 bp on average. After clarification by centrifugation, sonicated extracts were adjusted to the conditions of RIPA buffer (10mM Tris pH 8.0, 1mM EDTA pH 8.0, 1% Triton x100, 0.1% SDS, 200mM NaCl, Na Deoxycholate 0.1%). 2 µg of anti-RNAPII (ab5408), anti-TOP1 (ab109374), anti-H3K4me3 (millipore, 04-745) were mixed with 30 µl of Dynabeads Protein A (Invitrogen) and incubated at 4°C for 6 h with rotation. Chromatin from 2.5×10^6 cells was added to the Protein A-antibody complexes and incubated overnight at 4°C with rotation. Immunoprecipitates were washed twice with RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% TritonX100, 0.1% Na-Deoxycholate, 0.1% SDS, 200mM NaCl); twice with RIPA buffer plus 300mM NaCl; once with LiCl buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP40, 0.5% Na-Deoxycholate); and twice with TE. The beads were then resuspended in 100 µl TE plus 0.25% SDS supplemented with proteinase K (500µg/ml, Thermo Fisher) and incubated overnight at 65ºC. The DNA was recovered from the eluate by phenol chloroform extraction followed by ethanol precipitation in the presence of 20 mg of GlycoBlue (Invitrogen) and dissolved in TE. DNA from ChIP was quantified with the Qubit dsDNA HS Assay Kit. Sequencing libraries were created according to the ThruPLEX DNA-seq kit protocol (Takara). Size selection was performed in the range of 200-700bp with AMPure XP beads and confirmed using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer. Libraries were pooled and sequenced using the NextSeq 500/550 High Output Kit v2.5 (Illumina). The sequencing run was Single End and Dual Index with 75 bp reads.