title: GSM7151688: ChIP-seq.BT549_SLUG-ChIP.scramble_input; Homo sapiens; ChIP-Seq
library_construction_protocol: RNA-seq: Total RNA was extracted using TRI-reagent and purified using the Direct-zol RNA MiniPrep Kit (Zymo). For KAT2B ChIP, MCF7ras+SS cells were harvested and double-crosslinked in suspension with 2mM DSG in PBS for 30 minutes at room temperature, washed with PBS twice and further crosslinked with 1% formaldehyde (FA) in PBS for 15 minutes at room temperature. Crosslinking was quenched by addition of glycine to final concentration of 125mM and incubated at room temperature for 10 min. For all other ChIPs, BT549 and MCF7ras+SS cells were subjected to crosslinking in 1% formaldehyde (FA) in PBS for 15 minutes at room temperature and followed by quenching with 125mM glycine for 10 minutes. Fixed cells were then subjected to standard ChIP lysis protocol to release chromatin. Released chromatin was sonicated to obtain fragments between 100-300bp, pre-cleared and used for IP with antibody-Dynabead complex. Captured fragments were subjected to several rounds of washes before elution with SDS buffer. RNA-seq: Equal amount of RNA preparations (extracted from equal number of cells) from each sample were subjected to ribosomal RNA depletion using NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (NEB Cat# E6310) and library construction using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB Cat# E7760), according to manufacturer's protocol. ChIP-seq: Eluted DNA and DNA from relevant input materials was purified and made into libraries using NEBNext Ultra II DNA kit (NEB Cat #: E7645). For both RNA-seq and ChIP-seq libraries, average fragment size was determined on Agilent High Sensitivity DNA and D1000 Screentape system (Agilent Cat #:5067-4626, 5067-5582), quantified using Qubit High Sensitivity DNA system (Life Tech Cat# Q32854), and pooled in equimolar. Pooled libraries were sequenced on the Illumina NextSeq or HiSeq platform.