GSM3670062: Cal27 H3K27ac ChIP seq; Homo sapiens; ChIP-Seq
Chromatin was sheared in diluted lysis buffer (1% SDS, 50mM Tris-HCl, 20mM EDTA, 1x protease inhibitor) to 200-500bp using a Covaris M220 Focused-Ultrasonicator with the following parameters: 10 minutes, peak incident power 75, duty factor 10%, 200 cycles/burst. 3ug antibody anti-H3K27Ac (Active Motif, #39133) was incubated with Dynabeads Protein G beads (Invitrogen, #10004D) for 4 hours. Beads were washed 3 times with 5mg/ml BSA, and incubated with 50% crosslinked chromatin (500ul) and 50% ChIP master mix (1% Triton-X, 0.1% DOC, 1X protease inhibitor, 1x TE) at 4°C overnight. 10% of the chromatin was not exposed to antibody and was used as control (input). Immunoprecipitated DNA was washed with RIPA buffer (50mM Hepes, 1% NP-40, 0.7% DOC, 0.5M LiCl, 1mM EDTA, 1x protease inhibitor) and eluted from the beads in elution buffer (10mM Tris, 1mM EDTA, 1% SDS). Along with input, immunoprecipitated DNA was reverse crosslinked at 65°C overnight, and purified via Phenol:Chloroform extraction DNA libraries were prepared with either the TruSeq ChiP Library Prep Kit (Illumina, #IP-202-1012) or the Accel-NGS 2S Plus DNA Library kit (Swift Bioscience, #21024) and sequenced using 75 bp single-end sequencing method on an Illumina Hi-seq 4000