CoBATCH for high-throughput single-cell epigenomic profiling

Abstract

An efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is so far lacking. Here we develop CoBATCH, combinatorial barcoding and targeted chromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions and Tn5 generates indexed chromatin fragments ready for the library preparation and sequencing. Importantly, through a combinatorial barcoding strategy, we are able to measure epigenomic features up to tens of thousands single cells per experiment. CoBATCH produces not only high signal-to-noise features, but also ~10,000 reads per cells, allowing for efficiently deciphering epigenetic heterogeneity of cell populations and subtypes and inferring developmental histories. Thus, obviating specialized device, CoBATCH is easily deployable for any laboratories in life science and medicine. MAIN TEXTSingle cell Transcriptomics 1, 2 and single cell Epigenomics 3-8 have seen great stride and invaluable utilities in revealing cell fate specification and population heterogeneity during developmental processes and pathological alterations.Development of efficient and robust single-cell ChIP-seq (Chromatin Immunoprecipitation in parallel with Sequencing) methods for profiling epigenetic landscape far lagged behind, despite numerous efforts and progress on low input cells have been made 9-13 . Here, we developed a versatile strategy, dubbed in situ ChIP for a small amount of materials and combinatorial barcoding and targeted chromatin release (CoBATCH) for single-cell level.For in situ ChIP with low-input cells, we placed the N terminal of Tn5 transposase to Protein A (PAT) for targeted tagmentation and the one-step sequencing library preparation, obviating DNA end repairing and adaptor ligation.Varying cell numbers of mouse embryonic stem cells (ESCs) were permeabilized and incubated with antibodies (H3K27ac, H3K27me3 and H3K4me3) before addition of PAT to tether Tn5 transposase to the specific histone marks or proteins of interest through protein A ( Figure 1A). Targeted tagmentation and indexed fragment release were performed, followed by PCR enrichment for the sequencing ready library in one tube without need of DNA extraction and purification. We acquired high quality in situ ChIP signals for all three histone marks as few as 100 cells, as demonstrated by specific inspection of a few positive peak regions defined by public data from ENCODE (Supplementary Figure 1A-C). Notably, due to the high signal-to-noise advantage of this approach, the sequencing depth of the unique non-duplicated reads of ~5M generated highly reproducible data as demonstrated by peak intersection. Highly enriched H3K27me3 signals, a repressive mark preferentially found in regions of lower chromatin accessibility 14 , confirmed that in situ ChIP method was compatible not only to active histone marks, largely overlapping with ope...

Unraveling Heterogeneity in Transcriptome and Its Regulation Through Single-Cell Multi-Omics Technologies

“…A variety of single-cell assays are developed to map the chromatin accessibility landscapes, relying on the differential enzymatic access to the chromatin with varying degrees of openness (Buenrostro et al, 2015;Cusanovich et al, 2015;Jin et al, 2015;Pott, 2017). Droplet-based single-cell ChIP-Seq approaches are developed to map the genetic regions bound by proteins of interest (Rotem et al, 2015;Grosselin et al, 2019;Wang et al, 2019). In spite of the significant improvements to genomic coverage over the years, applications for scChIP-Seq thus far are mostly limited to profiling histone modifications, but not transcription factors which present lesser prevalence in terms of genomic binding.…”

Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions

“…To accurately align the multi-layered omics information within the same cell, we developed CoTECH, a method for a combinatorial indexing-based single-cell co-assay for transcriptome and chromatin occupancy. Profiling chromatin parts in CoTECH is based on our previously well-established CoBATCH (Wang et al, 2019) with a few modifications that allow simultaneously measuring mRNA by the following steps (Figures 1 and S1; Table S1; STAR Methods): (i) Single cells on concanavalin A coated beads are permeabilized and incubated with primary and secondary antibodies. (ii) Cells bound with the antibody are distributed into a 96-well plate with protein A-fused Tn5 transposon (PAT) assembled with a unique combination of T5 and T7 barcodes in each well.…”

“…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 profiling of multi-dimensional chromatin states have been developed, such as various scChIP-seq techniques for DNA-binding proteins and histone modifications Kaya-Okur et al, 2019;Ku et al, 2019;Rotem et al, 2015;Wang et al, 2019), scATAC-seq for chromatin accessibility (Buenrostro et al, 2015), MNase-seq for nucleosome positioning (Lai et al, 2018), scBS-seq for DNA methylome (Guo et al, 2015) and scHiC for higher-order chromatin structure (Nagano et al, 2013). Although these methods measure multiple modalities of single cells, each provides only specific layers of cellular heterogeneities.…”

“…Here, we develop a high-throughput method for single-cell joint detection of chromatin occupancy and transcriptome. This approach, named CoTECH (combined assay of transcriptome and enriched chromatin binding), adopts a combinatorial indexing strategy (Cusanovich et al, 2015;Wang et al, 2019) to enrich chromatin fragments of interest as reported in CoBATCH in combination with a modified Smart-seq2 procedure (Picelli et al, 2014) to simultaneously capture the 3' mRNA profiles in the same single cells. To demonstrate the utility of CoTECH, we used it to study the relationship between multiple histone modifications and gene expression in mouse embryonic stem cells (mESCs) and the regulatory basis of endothelial-to-hematopoietic transition (EHT) in two waves of hematopoietic cells.…”

Single-cell transcriptomics uncover distinct innate and adaptive cell subsets during tissue homeostasis and regeneration

“…85 Nevertheless, scRNA-Seq can only In addition to the transcriptome and proteome levels, cellular heterogeneity can also be examined at the genomic and epigenetic levels. For instances, methylation and histone modification patterns of individual cells can be profiled by methods such as scBS-Seq 89 or scM&Tseq, 90 and CUT&Tag 91 or CoBATCH, 92 respectively. Genomewide chromatin accessibility of individual cells can also be assessed by single-cell Assay for Transposase Accessible Chromatin using sequencing.…”