ChIP-seq of RNA polymerase II using pan-pol II and S2-phosphorylated C-terminal domain antibodies in mouse embryonic stem cells

ABSTRACT: Our proteomics data (PXD030253) indicate RNA polymerase II is co-localized with Aldh18a1. To confirm this observation using an orthogonal method we performed ChIP-seq experiments to identify the binding patterns of RNA polymerase II in general, and RNA polymerase II with S2-phosphorylated CTD. We also performed Cut&Run using Aldh18a1 antibody which is submitted separately. To do a ChIP-seq, mES cells (46C) were treated with Accutase to be detached. The cells were fixed by 1.5% formaldehyde in PBS for 15min. Chromatin was fragmented by sonication and sheared to < 500 bp fragments. The cell debris were removed by spinning. The sheared chromatin was treated with 10 ug antibodies (pan-Pol II Bethyl A304-405A, or S2P-Pol II Bethyl A304-407A) overnight at 4 °C. Dynabeads protein A beads (40ul) were added to the samples and the samples were rotated 2-3 hours to perform the immunoprecipitation. The beads were washed 5 times. Chromatin was eluted in a denaturing solution, and proteins were digested using proteinase K. DNA was purified by SPRI beads. The ChIP libraries were prepared using NEBNext Ultra II DNA library prep according to the manufacturer manual. The libraries were sequenced using 150nt reads on paired-end mode by HiSeq4000. Reads were trimmed, aligned to the mouse genome (mm10) using Bowtie2, and duplicated reads were removed with samtools. The ChIP quality was evaluated by cross-correlation using the “SPP'' tool as suggested by ENCODE ChIP-seq guidelines. Peak calling was performed using MACS2.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Mahmoud-reza Rafiee 

PROVIDER: E-MTAB-11510 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress