GSM7767228: 231K, INPUT, Rep A; Homo sapiens; ChIP-Seq
Cells were washed twice with cold PBS and lysed (Buffer B; PN 520237, Covaris). Nuclei were washed (Buffer C; PN 520237, Covaris) and resuspend in sonication buffer (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% N-lauroylsarcosine sodium salt and fresh 0.1% Na-deoxycholate and proteinase inhibitors). Chromatin was fragmented by sonication with a Bioruptor to an average size of 100-400 bp and brought to 1% TritonX-100. A 50 μL aliquot was saved as input, whereas 500 μL each was incubated with 5 μg antibody, overnight at 4°C. Protein A magnetic beads (16-661, Merk Millipore) were washed three times with 0.5% BSA in PBS and added for 5 h at 4°C. DNA-protein complex was recovered, washed and eluted overnight at 65°C (GeneJET PCR purification; K0701, Thermo Fisher Scientific). Library preparation from 5 ng purified chromatin was performed using NebNext Ultra II DNA Library Prep with magnetic purification beads (E7103S, NewEngland Biolabs) and multiplex oligonucleotides (Dual Index Primers; E7600S, NewEngland Biolabs), followed by adaptor ligation according to the manufacturer's protocol. Quality controls of amplified chromatin fragments were performed on Bioanalyzer 2100 (Agilent Technologies) and Qubit V4 (Thermo Fisher Scientific). Next-generation sequencing was performed on NextSeq2000 (Illumina) with the P2 reagents kit V3 (100 cycles; Illumina)