GSM5463056: ChIPseq D51A Input rep1; Homo sapiens; ChIP-Seq
Native ChIP was performed as previously described using with 5×106 cells. The cells were trypsinized to release them from the culture dishes, washed, and lysed using Hypotonic Lysis Buffer [50 mM Tris-HCl pH 7.4, 1 mM CaCl2, 0.2% Triton X-100, 250 nM ADP-HPD (Sigma-Aldrich, A0627, a PARG inhibitor), 10 μM PJ34 (Enzo Life Sciences, ALX-270-289, a PARP inhibitor), and protease inhibitor cocktail (Roche)] with micrococcal nuclease for 5 min at 37 °C to recover mono- to tri-nucleosomes.  Nuclei were lysed by brief sonication and dialyzed into RIPA Buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100) for 2 hr at 4 °C.  Soluble material (30 µg) was incubated with 2.5 μg of H2BK12ac antibody bound to 25 μL protein A Dynabeads (Invitrogen) and incubated overnight at 4 °C, with 5% reserved as input.  Magnetic beads were washed as follows: 3x RIPA buffer, 2x RIPA buffer + 300 mM NaCl, 2x LiCl Buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), 1x TE + 50 mM NaCl.  Chromatin was eluted and treated with Proteinase K.  The ChIP'd DNA was purified and eluted in water. ChIP-seq libraries were prepared from 5 ng of ChIP'd DNA following the Illumina TruSeq protocol.  The quality of the libraries was assessed using a D1000 ScreenTape on a 2200 TapeStation (Agilent) and quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher).  Libraries with unique adaptor barcodes were multiplexed and sequenced on an Illumina NextSeq 500 (paired-end, 33 base pair reads).  The ChIP-seq libraries were sequenced to an average depth of ~35 million unique aligned reads per condition.  Two independent biological replicates were used.