CoTECH for single-cell joint detection of transcriptome and chromatin occupancy

Here we present CoTECH, a high-throughput co-aasay that measures chromatin occupancy and transcriptome in single cells. The CoTECH method adopts a combinatorial indexing strategy to enrich chromatin fragments of interest as reported in CoBATCH in combination with a modied Smart-seq2 procedure to simultaneously capture the 3’ mRNA proles in the same single cells. The whole experimental procedure can be handled within three days.The CoTECH acquires data quality of 1000-9000 unique mapped reads (DNA partition) and 1500-4000 expressed genes (RNA partition) per cell. Experimentally linking chromatin occupancy to transcriptional outputs and inferred molecular association between multimodal omics datasets made possible by CoTECH enables reconstructions of higher dimensional epigenomic landscape, providing new insights into epigenome-centric gene regulation and cellular heterogeneity in many biological processes. This step-by-step protocol is related to the publication “Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions” in Nature Methods.


Introduction
Single-cell transcriptome sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity in physiological and pathological biological processes [1][2][3][4][5][6][7] . The transcriptome and cell identities of different cell types/states with the same DNA are determined by multilayers of epigenetic information. Recently, technologies for single-cell pro ling of multi-dimensional chromatin states have been developed, such as various scChIP-seq techniques for DNA-binding proteins and histone modi cations [8][9][10][11][12] , scATAC-seq for chromatin accessibility 13 , MNase-seq for nucleosome positioning 14 , scBS-seq for DNA methylome 15 and scHiC for higher-order chromatin structure 16 . Although these methods measure multiple modalities of single cells, each provides only speci c layers of cellular heterogeneities. To build connections across these layers in single cells, innovative computational platforms emerge to integrate unpaired single-cell omics datasets and project different molecular information into a common latent space 17,18 . However, existing strategies require priori knowledge-based correspondence across multimodal omics datasets from different experiments, limiting the ability to reconstruct an accurate view of functional relationship between different modalities of epigenomic features and gene expression as well as the crosstalk between different epigenetic layers.
More recently, unlike those pro ling molecular layers one at a time, new approaches have been developed to experimentally link the transcriptome and epigenome by simultaneously measuring multi-omics in the same single cells, making it possible to precisely analyze single-cell-resolved epigenomic regulation of gene expression and cell fate decisions. For example, several various single-cell co-assays (combined scATAC-and scRNA-seq) for joint analysis of accessible chromatin and gene expression have been developed, permitting inference of the correlation between cis-regulatory elements and putative target genes [19][20][21][22] .
Apart from chromatin accessibility, other important molecular layers of the epigenome are covalent modi cations to histones and chromatin occupancy of DNA binding proteins, providing a critical guidance for determining transcriptional outcomes 23 . Two elegant studies demonstrated the proof-ofconcept application of simultaneously quantifying of protein-DNA interactions and transcriptome in single cells by scDam&T-seq and scCC 24,25 . Both methods rely on a transgene expressing transcription factors or chromatin binding proteins tethered to Escherichia coli DNA adenine methyltransferase (Dam) or piggyBac transposase for scDam&T-seq and scCC, respectively, limiting the likelihood of wide adoption and the potential implementations, in particular for studying clinical human samples. Moreover, the same strategy cannot measure various histone modi cations in single cells. Here, we develop a highthroughput method for single-cell joint detection of chromatin occupancy and transcriptome. This 13. Tagmentation: Incubate the plate at 25°C for 1 h in a thermal cycler. The reaction system is gently mixed once after 30-min incubation.
14. Add 10 µl 40 mM EDTA to each well and mix well. Incubate the plate at 4°C for 15 min. 15. Add 1 µl 250 mM MgCl 2 to each well and incubate the plate at 4°C for 5 min. Attention: RT lysis Buffer should be prepared before the experiment. One kind of barcoded oligo dT primer is added to one well individually, which makes all 96-wells carrying different barcoded oligo dT. Carefully note the corresponding PAT-barcode and oligo dT-barcode for each well.
18. Incubate the plate at 55°C for 5 min and immediately put the plate onto a cold block to cool down.  37. Add 1 µl 1.8% TX-100 to each well to quench SDS, and incubate the plate at 55°C for 5 min.