GSM7036227: NALM6_polII_rep2; Homo sapiens; ChIP-Seq
RNA polymerase II ChIP-seq data were generated by first fixing 20 million Nalm6 cells in 1% formaldehyde (diluted from sigma F87750) at room temp for 10 minutes. Crosslinking was stopped with the addition of 2.5M glycine to a concentration of 0.125M, and the cells were then washed in ice-cold PBS. 5mg anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] (ab5408, lot: GR3264797-1) was prebound to 200ul of protein G dynabeads (invitrogen 10003D) overnight in 0.5% BSA in PBS. Fixed cell pellets (20M cells) were suspended in 1ml Farnham lysis buffer (5mM PIPES pH 8, 85mM KCl, 0.5% NP40, 1x protease inhibitors (Roche 11836170001)) and passed through a 18G needle 10x. Nuclei were pelleted and resuspended in 275ul of RIPA buffer (1x PBS, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1x protease inhibitors) and sonicated on high power in 1.5ml tubes for 25 minutes (30s on/ 30s off) using a Diagenode Bioruptor Plus. 5% Input samples were taken from sonicated material and the remaining sonicated material was added to the pre-bound antibody/protein G beads to rotate overnight at 4C. The next day the supernatant was discarded, and the beads were washed 5x with ice cold LiCl buffer (100mM Tris pH 7.5, 500mM LiCL, 1% NP40, 1% sodium deoxycholate) and 1x with ice cold TE buffer (10mM Tris pH 7.5, 1mM EDTA). Samples were eluted from the washed beads using room temperature IP elution buffer (1% SDS, 0.1 M NaHCO3) at 65C for 1hr, vortexing every 15 minutes. The elution was then incubated at 65C overnight to reverse crosslinks. The next day DNA was purified using the QIAquick PCR purification kit (Qiagen 28104). DNA quantification was performed using the PicoGreen assay (Molecular Probes, Eugene, OR, P-7581). Sequencing libraries were generated from ChIP and input DNA by using the KAPA Hyper Prep kit (Roche, Basel, Switzerland, # 7962363001) according to the included manufacturer's specifications, and quality was determined by using the Agilent TapeStation with D1000 screentape. Then, >50M 50-bp paired-end reads per sample were generated on the NovaSeq 6000.