iPSC H3K27Ac: Sample e8d0daf3-5f47-44eb-afdd-f060c0d8a3f9
For H3K27ac, 2 x 106 fixed cells were lysed in 60 µl of MAGnify™ Chromatin Immunoprecipitation System Lysis Buffer (Thermo Scientific) and sonicated using Bioruptor 200 (Diagenode) for 35-45 min of 30 sec on/30 sec off cycles. H3K27ac antibodies (Abcam ab4729, lots GR183922-2 (1.75 µg) or GR184333-2 (1 µg)) were coupled for 2 hours to ProteinG Dynabeads (Thermo Scientific), and used for overnight chromatin immunoprecipitation in IP buffer (1% Triton-X, 0.1% DOC, 1x TE, 1x Roche Complete Proteinase Inhibitor tablets (RCPI)). Beads were washed five times with washing buffer (50 mM Hepes pH 8, 1% NP-40, 0.7% DOC, 0.5M LiCl, 1mM EDTA and 1x RCPI) and once with TE buffer. For transcription factors, 1-2 x 107 cells were lysed in 300 µl RIPA buffer (1xPBS, 1% NP-40, 0.5% DOC, 0.1% SDS, RCPI) and sonicated for 70-80 min with instrument and setting as above. Five µg of SRF antibody (Santa Cruz Biotechnology, sc-335x, lot I1014) were incubated with Dynabeads for 2 hours and washed with BSA 0.5% in PBS. Chromatin was diluted to 1 ml of RIPA buffer and added to the beads for overnight IP. Five washes were performed with washing buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% DOC and 1x RCPI), followed by one wash with TE. DNA was eluted and reverse crosslinked overnight in elution buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 1% SDS) at 65˚C. DNA was purified using Qiagen MinElute PCR Purification kit, quantified by Qubit (Thermo Scientific) and submitted to library preparation and barcoding using KAPA Hyper Library preparation kit (KAPA Biosystems). Libraries were sequenced on an Illumina HiSeq2500 or a HiSeq4000 to an average of 35 M 100 bp paired-end reads per sample.
alignment_software
BWA-MEM