GSM9290581: H661-K4K119-R2; Homo sapiens; ChIP-Seq
For each reChIP 1x10^6 single-crosslinked NCI-H661 cells were used. Pellets of single-crosslinked NCI-H661 cells were washed twice with 1mL NP buffer (10mM Tris-HCl, 1M sorbitol, 50mM NaCl, 5mM MgCl2, 1mM CaCl2, 0.1% IGEPAL CA-630, 0.1% Tween-20 in H2O freshly supplemented 1x protease inhibitor tablet) on ice. Nuclei were pooled and resuspended in 800µL ChIP buffer (as for ChIP), then left to incubate on ice for at least 5 minutes. Chromatin was transfered to a milliTUBE AFA Snap Fibre sonication vial (Covaris, #520135) and sheared on a Covaris ME220 instrument for 20mins (4C, 75% power, 15% duty factor, 1000 cycles) and debris were pelleted at 12,000xg for 15 mins at 4°C. The supernatant containing chromatin was pre-cleared by adding 20µL washed Protein-A dynabeads resuspended in ChIP dilution buffer: 67mM Tris-HCl, 100mM NaCl, 5mM EDTA, 0.33% SDS, 1.67% Triton-X) and incubated at 4°C with rotation for at least 3 hours. 10µL of the pre-cleared chromatin was set aside as 5% input. For each reChIP, 200µL of pre-cleared chromatin (1x106 cells) was made up to 500µL in a dilution buffer to get a final concentration of 67mM Tris-HCl, 100mM NaCl, 5mM EDTA, 0.33% SDS, 1.67% Triton-X, 0.1μM PMSF, 1x protease inhibitors (reChIP buffer). 20µL Protein-A dynabeads were pre-incubated (as for ChIP) with either 0.5µg H3K4me3 (CST, #9751) or H2AK119ub (CST, #8240) antibodies or Rabbit IgG (Invitrogen, #02-6102), then resuspended in the 500µL diluted chromatin. The first immunoprecipitation was performed overnight at 4°C with rotation, antibody-chromatin complexes were then washed three times in 500µL low-salt buffer (as for ChIP), three times in 500µL high-salt buffer (as for ChIP), twice in 200µL LiCl buffer (as for ChIP) and twice in 500µL 10mM TrisCl pH7.5 on ice. Complexes were eluted in 100µL ChIP elution buffer (as for ChIP) supplemented with 0.1μM PMSF and 1x protease inhibitors for 37°C for 30 mins on a thermomixer at 300RPM, of which 10µL were put aside as a 10% single ChIP control. To dilute the SDS, the eluate volume was increased to 300µL with reChIP buffer and purified using Amicon Ultra-0.5ml 3kDa filter columns (Millipore, #UFC5003) according to the manufacturer's instructions, with chromatin being additionally washed twice with 400µL ChIP dilution buffer. Purified chromatin was made up to 500µL in reChIP buffer for the second immunoprecipitation, which was performed using the alternate antibody or IgG control bead conjugates overnight at 4°C with rotation. Complexes were washed (as for ChIP) and resuspended in 100µL ChIP elution buffer. Samples were treated with 2µL RNase A and 2µL Proteinase K, then reversed crosslinked (as for ChIP). DNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB) and eluted in 25µL H2O for qPCR and library preparation for sequencing. reChIP libraries were prepared using the NEBNext UltraII DNA Library Preparation Kit (NEB) according to the manufacturer's instruction. During library preparation the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-4) were used to enable library multiplexing. Individual and pooled libraries were quantified and quality controlled using the dsDNA high sensitivity (HS) Qubit assay (Invitrogen) on the Qubit 3.0 fluorometer (Invitrogen) and HSD1000 High Sensitivity DNA kit (Agilent) on the TapeStation 4150 (Agilent). Libraries were pooled and sequenced on the NextSeq2000 platform in a single-end configurations with 100bp read lengths.