GSM8004767: SW480_H4K12ac_prop_ChIP_2; Homo sapiens; ChIP-Seq
Cells were trypsinized and cross-linked with 1% formaldehyde (EMD Millipore) for 10 min at RT and quenched with 2 M glycine (Thermo Fisher Scientific) for 5 min at room temperature. Chromatin corresponding to 10 million cells for histone modifications was sheared with SFX250 Sonifier (Branson). The lysates were transferred to Diagenode tubes, sonicated 16 rounds of 30 seconds on 30 seconds off, vortexing every 4th round. Protein G beads (80uL) and lysates were washed with RIPA buffer and immunoprecipitated with antibodies targeting given Kacyl marks. 5 μg for each condition, at 4 °C overnight on a nutator. For the input sample, 200 μl of sheared nuclear lysate was removed and stored overnight at 4 °C. ChIP-DNA and input was reverse crosslinked by incubating overnight at 65 °C in 1% SDS, 1x TE buffer. On the third day, ChIP-DNA was treated with RNase A (Qiagen) and Proteinase K (ThermoFisher Scientific) and then purified using Qiagen QIAquick purification columns. The ChIP-DNA samples were end repaired using End-It DNA End Repair Kit (Lucigen) and A-tailed using Klenow Fragment and dATP (New England Biolabs). Illumina TruSeq adapters (Illumina) were ligated and libraries size-selected (200 - 400 bp) by gel extraction before PCR amplification. The purified libraries were subjected to paired-end sequencing on the Illumina HiSeq 4000/Novaseq 6000 SP to obtain an average of approximately 30 - 35 million uniquely mapped reads for each sample