Highlights d Development of an immunoprecipitation-free single-cell ChIP-seq technology d CoBATCH acquires 12,000 usable reads per cell with extremely low background d CoBATCH reveals multi-layer epigenetic heterogeneity during cell lineage diversification

No 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...