HiCAR: a robust and sensitive multi-omic co-assay for simultaneous measurement of transcriptome, chromatin accessibility, and cis-regulatory chromatin contacts

High-order chromatin organization plays a central role in regulating spatial-temporal gene expression by facilitating or constraining the interactions between cis-regulatory elements (cREs). cREs are often the accessible DNA sequences and can be identified at genome-wide scale with assays such as ATAC-seq, DHS-seq, and FAIRE-seq. However, it remains technically challenging to comprehensively identify the long-range interactions that occur between cREs, especially using low-input cells and in a cost effective manner. Here, we report HiCAR, Hi gh-throughput C hromosome conformation capture on A ccessible DNA with m R NA-seq co-assay, which enables simultaneous mapping of chromatin accessibility and cRE anchored chromatin contacts. Notably, using the same input material, HiCAR also yields high-quality transcriptome data that represents the functional outputs of chromatin accessibility and interaction. Unlike immunoprecipitation-based methods such as HiChIP, PLAC-seq, and ChIA-PET, HiCAR does not require target-specific antibodies and thus can capture cis-regulatory contacts anchored on the accessible DNA regions associated with multiple epigenetic modifications and transcription factor binding. We compared HiCAR to another technology designed to capture interactions between accessible chromatin regions, called Trac-looping, and found that HiCAR yielded much more informative long-range cis- reads at similar sequencing depth, and requires far fewer cells as input. We applied HiCAR to H1 human embryonic stem cells (hESC) and identified 46,792 open-chromatin anchored loops at 5Kb resolution. Interestingly, we found that the poised cREs form extensive and significant chromatin interactions comparable to the active cREs. We further showed that the spatial interactive activity of cREs do not correlate with their transcriptional activity, enhancer activity, and chromatin accessibility. Additionally, we identified 2,096 super interactive regulatory (SINTER) loci showing abnormally high levels of chromatin interactivity and associated with unique epigenetic features. In summary, HiCAR is a robust, sensitive, and cost effective multi-omics coassay that can be used to study chromatin structure and function as well as gene expression.