GSE162819
HiCAR analysis of human embryonic stem cell differentiation reveals principles of cis-regulatory chromatin reorganization controlling developmental gene expression
we report a novel method, Hi-C on Accessible Regulatory DNA (HiCAR), which leverages principles of ATAC-seq and high-throughput 3C-assay for genome-wide profiling of chromatin accessibility and open chromatin anchored cRE interactions. Overall design: cross-linked cells were treated with Tn5 transposase that were assembled with an engineered DNA adaptor . The Tn5 adaptor contains a Mosaic End (ME) sequence for Tn5 recognition as well as a single-stranded flanking sequence that can be ligated to the CviQI-digested DNA fragment with a splint oligo. Next, restriction enzyme digestion was performed using the 4-base cutter CviQI, followed by in situ proximity ligation to ligate the Tn5 adaptor to the spatially proximal genomic DNA. After in situ ligation, cross-links were reversed, and the purified DNA was digested by another 4-base cutter NlaIII, followed by circularization and PCR amplification to generate HiCAR DNA libraries for Next-Generation-Sequencing (NGS)