cervical adenocarcinoma epithelial cell line, HeLa
expressing
GFP
chip antibody
GFP custom antibody (Michiel Vermeulen's lab)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-Seq HeLa Kyoto wild type (WT), HeLa EMSY GFP, HeLa GATAD1 GFP and HeLa EMSY GFP with knockdown of ZNF131 were seeded in 15 cm dishes and 24 hours later were formaldehyde crosslinked for 10 minutes. Chromatin obtained from the cells after sonication was subjected to overnight IP using GFP antibody (Abcam, ab290) followed by extensive washes and decrosslinking. DNA was purified using the Qiagen PCR clean-up kit and the bound DNA was analysed by qPCR using the desired primers. ChIP-Seq was carried out by conventional ChIP followed by end repair of 15 ng (for protein-GFP fusions) or 30 ng (for the histone modifications) enriched DNA as measured by Qubit fluorometer using the Quant-iT dsDNA HS Assay Kit from (Invitrogen, Q32851). Adaptors were ligated to DNA fragments, which were subsequently size selected (∼300 base pair [bp]). The adaptor-modified DNA fragments were subjected to limited PCR amplification (14 cycles) and quality control was made by qPCR (primers sequences are available upon request), as well as by running the PCR products on a Bioanalyzer (BioRad). Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) according to standard protocols of the manufacturer (Illumina).