GSM4730426: H3K4me1_SFTregD2; Homo sapiens; ChIP-Seq
For each sample, cells were crosslinked with 2% formaldehyde and crosslinking was stopped by adding 0.2 M glycine. Nuclei were isolated in 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, and 1% Triton X-100 and lysed in 20 mM Tris (pH 7.5), 150mMNaCl, 2mMEDTA, 1% NP-40, 0.3% SDS. Lysates were sheared using Covaris microTUBE (duty cycle 20%, intensity 3, 200 cycles per burst, 60-s cycle time, eight cycles) and diluted in 20 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% X-100. Sheared DNA was incubated overnight with anti-histone H3 (mono methyl K4) antibody (ab8895; Abcam) pre-coupled to protein A/G magnetic beads. Cells were washed and crosslinking was reversed by adding 1% SDS, 100mM NaHCO3, 200mM NaCl, and 300 µg/ml proteinase K. DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research), end-repair, a-tailing, and ligation of sequence adaptors was done using Truseq nano DNA sample preparation kit (Illumina). Samples were PCR amplified, checked for the proper size range and absence of adaptor dimers, and barcoded libraries were sequenced 75 bp single-end on an Illumina NextSeq500 sequencer.