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)

本研究报道了一种全新方法——基于可及调控DNA的Hi-C技术（Hi-C on Accessible Regulatory DNA，简称HiCAR），该方法结合转座酶可及性测序（ATAC-seq）与高通量染色质构象捕获试验（3C-assay）的原理，可实现染色质可及性与开放染色质锚定的顺式调控元件（cRE）相互作用的全基因组图谱分析。整体实验设计：对经交联固定的细胞，使用组装有工程化DNA接头的Tn5转座酶进行处理。该Tn5接头包含用于Tn5识别的镶嵌末端（Mosaic End，ME）序列，以及可通过夹板寡核苷酸连接至经CviQI酶切的DNA片段的单链侧翼序列。随后，采用4碱基识别位点限制性内切酶CviQI进行限制性酶切，继而开展原位邻近连接反应，将Tn5接头与空间邻近的基因组DNA进行连接。原位连接完成后，逆转交联反应，将纯化后的DNA使用另一4碱基识别位点限制性内切酶NlaIII进行酶切，随后通过环化与PCR扩增生成用于下一代测序（Next-Generation-Sequencing，NGS）的HiCAR DNA文库。