ChIP-Seq for RNA Polymerase II in RASMC under PDGF stimulation and JQ1 treatment at 2 hours
cell line
RASMC
tissue
Rat aortic smooth muscle cell
input
RASMC_WCE_PDGF_JQ1_2H_NEW
treatment
JQ1 (500nM) pre-treatment for 3 hours followed by PDGF (25ng/ml) for 2 hours
barcode
GATCAG
antibody
RNA Pol II
antibody maker
Biolegend
antibody catalog number
920102
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP was performed as described previously (PMID: 27425608). Briefly, cells were fixed with 1% formaldehyde for 10min, quenched with 0.125M glycine for 5min and washed with ice cold PBS containing protease inhibitor. Cell pellets were snap frozen in liquid nitrogen and saved in -80oC. Cells were lysed in lysis buffer as described here PMID: 27425608 and chromatin was sheared using Diagenode Bioruptor plus (16 min, 30 sec on/30 sec off, setting high). Immunoprecipitation was performed using 25 million cells for each H3k27Ac (Abcam, ab4729, 5µg per IP) and RNA Pol II (Biolegend 920102 Clone 8WG16, 5µl per IP). 75 million cells and 5µg antibody was used for Brd4 IP (Bethyl A301-985A). IP Samples were reverse cross linked and DNA was purified using phenol:chloroform:isoamyl alcohol. Purified DNA samples were sent to WIGTC for NGS library preparation and sequencing. TruSeq ChIP library preparation kit was used according to manufacturer's instructions (https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-chip.html) ChIP-Seq with single end 40bp sequencing