GSE99889
Genome-wide maps of HIC1 binding sites in human induced regulatory T (iTreg) cells after 72h post polarization
CD4+ T cells were cultured in iTreg cell polarizing condition for 72h. ChIP was performed as previously described (Tripathi et al., Cell Reprot 2017) with slight modifications. The cells were subjected to sonication using Bioruptor® Pico sonication device (Diagenode) to attain 100–500 bp chromatin fragments size. A total of 250 ug of sonicated chromatin fragments were incubated with 10ug of HIC-1 antibody (Santa Cruz, sc-271499) and incubated for crosslinking with magnetic beads (no. 11201D, Dynabeads® M-280 Sheep Anti-Mouse IgG, Dynal Biotech, Invitrogen). The crosslink samples were reversed at 65oC overnight and precipitated DNA was treated with Rnase A and Proteinase K and purified with QIAquick PCR purifica-tion kit (QIAGEN). The DNA libraries were prepared as per the guidelines from Illumina by Fasteris Life Sciences (Plan-les-Ouates, Switzerland). Input DNA was sequenced and used as a control. DNA libraries were sequenced on Illumina HiSeq2500 producing from 25 to 35 million reads per sample. The 50 nucleotide reads were aligned to hg19 build of human reference genome using bowtie2 (ver-sion 2.2.9). Uniquely mapped reads were retained (~20-25 million reads per sample) for further analysis. HIC-1 binding sites relative to input DNA were identified using MACS2 (version 2.1.1.20160309) with the default parameter settings. De novo motifs from HIC-1 ChIP-seq peaks were discovered using Homer (version 4.8). The binding sites were annotated in terms of genomic annotations using the “annotatePeaks.pl” script supplied as part of Homer. HIC-1 motif locations in ChIP-Seq peaks were scanned using Homer with default parameters (with the addition of –local argument). Overall design: Identification of genome wide HIC1 binding sites  in human induced regulatory T (iTreg) cells at 72h post differentiation from naive CD4+ T cells.