Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells

概要

〔目的(Purpose)〕
Recent technological advances have enabled massively parallel chromatin profiling with single-cell Assay for Transposase Accessible Chromatin by sequencing (scATAC-sea) in thousands of individual cells. Here, we aimed to extend these approaches with the addition of multiple modalities to complement scATAC-seq and developed ATAC with Select Antigen Profiling by sequencing. ASAP-seq. a tool to simultaneously profile accessible chromatin and protein levels in thousands of single cells.

〔方法ならびに成績(Methods/Results)〕
Our approach pairs sparse scATAC-seq data generated on the 10X Chromium (10X Genomics) with robust detection of hundreds of protein markers and optional capture of mitochondrial DNA tatDNA) for clonal tracking, thus concomitantly capturing three distinct modalities in single cells. Importantly, ASAP-seq uses a novel bridging approach that repurposes antibody: oligo conjugates designed for existing technologies that pair protein measurements with single cell RNA-seq. This bridging approach introduces a unique 'UBI, which serves as a surrogate for the 'UMI, allowing for quantitative interpretation of protein tag abundances when using commercially available TotalSeq-A (BioLegend) oligonucleotide-antibody conjugates.

By benchmarking the assay with the gold-standard ‘species mix' experiment, we observed very low cross­ species contamination background, demonstrating specificity of protein detection in the assay.
Moreover, we found that there was no noticeable decrease in library complexity of the chromatin accessibility data, suggesting that the ATAC quality was comparable to standard scATAC-seq. Extending this to human PBMCs, we demonstrated staining of 9 major immune cell markers (TBNK) with cell-type specific specificity, which correlated well with imputed chromatin accessibility “gene activity scores”, suggesting that the staining was specific.

We next demonstrated proof-of-principle of the utility of ASAP-sea in CRISPR screening experiments (Perturb-ASAP) by encoding perturbation identities with protein combinatorial barcoding and applied this workflow lo dissect perturbation-specific epigenomic changes that occur during primary human CD4 T cell stimulation in vitro. This integrated approach allowed for unbiased discovery of context-dependent coding and non-coding gene regulation modules, exemplified by perturbation-specific chromatin and protein changes that occurred at the IL2RA locus.

Lastly, we demonstrate the unique compatibility of ASAPタンイツサイボウレベルニオケルオープンク ロマチンリョウイキカイセキ、イデンシハツゲンジョウホウナラビニタンパクシッティリョウノドウジソクテイニカンスルカクチョウカノウナタソウテキプロファイリングシュホウノカイハッ-seti with intracellular epitope staining of markers such as Granzyme B, Perforin. CTLA-4 and Ki-67 and
validated their specificity using CRISPR knockout experiments. Therefore, ASAP-seq offers new possibilities of defining nuanced cell states in single-cell genomics assays with precise quantitation of both surface and intracellular epitopes.

〔総括(Conclusion)〕
We established a novel method termed ASAP-seq that uniquely enables the simultaneous Quantitation of chromatin accessibility and protein epitopes in single-cells. We benchmarked ASAP-sea its utility in enabling unbiased discoveries through profiling of primary tissues and genomic perturbations. In total, our methodological approach and analyses demonstrate ASAP-sea as modular and powerful tools for charting the complex interplay of gene and regulatory layers in single cells.