Chromatin immunoprecipitation, ChIP-sequencing and data analysis

Chromatin immunoprecipitation was performed essentially as described previously (33). Cells were cross-linked with 1% formaldehyde for 15 minutes (for KLF10 ChIP) or 10 minutes (for H3K9Ac, H3K27Ac and HDAC1). Prior to library preparation, DNA samples were sonicated a second time using a Bioruptor® Pico (Diagenode) to ensure fragment sizes of approximately ≤ 300 bp. Fragmented DNA was used for library preparation using NEBNext Ultra DNA library preparation kit (New England Biolabs) according to the manufacturer’s protocol. IgG was used as a negative control. DNA was quantitated using the Qubit dsDNA HS assay and a Qubit® 2.0 Fluorimeter (Invitrogen). Three to five ng of DNA was used for library preparation. The fragment size of the libraries was analyzed using an Agilent Bioanalyzer 2100 (High Sensitivity DNA assay). Libraries were sequenced using HiSeq 2500 (Illumina) and sequence reads (single end, 50 bp) were aligned to the human reference genome (UCSC hg19) using Bowtie2 with default parameters (version 0.4) (34). Peak calling was performed using Model-based Analysis of ChIP-seq (MACS2) (version 2.1.0.20140616.0) on Galaxy Cistrome (35). The minimum FDR cutoff for peak detection (q-value) was set to 0.05. Bigwig files were generated using BamCoverage from deeptools (36). The bigwig data was visualized using Integrative Genomics Viewer (IGV, version 2.3.66) (37). Furthermore, bigwig and bed files were also used to generate aggregate profiles and relative enrichment of the ChIP regions over defined genomic locations using SitePro and CEAS tools, respectively, using the Galaxy Cistrome server. ChIP-seq and RNA-seq data have been deposited in Gene Expression Omnibus and are available under the accession number {"type":"entrez-geo","attrs":{"text":"GSE90567","term_id":"90567"}}GSE90567.