iPSC-CM NKX2-5: Sample c4f959d1-2b9a-4266-9dcc-7af4385828e0
For NKX2-5, 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 NKX2-5 antibody (Santa Cruz Biotechnology, sc-8697x, lot C0113) 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 (50 mM Hepes pH 8, 1% NP-40, 0.7% DOC, 0.5M LiCl, 1mM EDTA 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 to an average of 35 M 100 bp paired end reads per sample.
alignment_software
BWA-MEM