GSM5203352: GM23225_NPC_Pol2S2_unt_rep2; Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and RNAAPII-S2-DNA complexes were isolated with antibody. 2x10e7 cells were collected and cross-linked in 1% formaldehyde for 10 min at room temperature, then quenched by addition of 125mM Glycine. Nuclear lysates were prepared by sonication using a Branson 250 Sonifier. Clarified lysates were treated overnight at 4 °C with 5 µg anti-pol II CTD repeat YSPTSPS (phosphor S2) (Abcam ab5095) antibody. Protein-DNA complexes were captured on Protein A/G agarose beads (EMD Millipore) and eluted in 1% SDS TE buffer at 65 °C. Following cross-link reversal and purification, ChIP DNA sequencing libraries were prepared according to Illumina DNA sample kit instructions (Illumina). Libraries were sequenced on an Illumina HiSeq 4000. More than 300 million reads were obtained from each library in order to assure accurate quantification of RNAPII-S2 occupancy.