Single-nucleus chromatin accessibility and gene expression co-profiling by ISSAAC-seq

Multimodal profiling of different molecular layers from the same single cell enables more comprehensive characterization of cellular heterogeneity compared with conventional single-modality approaches. A key example is co-detection of chromatin accessibility and gene expression that offers the opportunity to investigate cell type-resolved gene regulatory mechanisms. Here we describe a sensitive and robust protocol for in situ sequencing hetero RNA-DNA-hybrid after assay for transposase-accessible chromatin using sequencing (ISSAAC-seq) for the concurrent measurement of chromatin accessibility and gene expression from the same single nucleus. The method begins with dual Tn5 tagging of open chromatin regions and the RNA-cDNA hybrid produced by reverse transcription that take place in bulk nuclei. Then, various single-nucleus isolation strategies, including plate and droplet barcoding-based approaches, can be used based on the experimental purpose of the user. The protocol is highly modular with a flexible throughput ranging from several hundreds to tens of thousands of nuclei. The generated data are of high quality in both modalities. The entire workflow can be finished within 1 or 2 days, and the procedures work on multiple different single-nucleus isolation and barcoding platforms.