scifi-ATAC-seq: massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing

Abstract

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow.

Keywords: ATAC-seq; Chromatin accessibility; Combinatorial fluidic indexing; Massive-scale; Single-cell.

Fig. 1

Scifi-ATAC-seq combines pre-indexing with droplet-based scATAC-seq. a Schematic of regular droplet-based 10X Genomics scATAC-seq experimental workflow. b Schematic of scifi-ATAC-seq experimental workflow. c Distributions of the proportion of Tn5 integration sites within the promoter regions, encompassing the 2-kb flanking regions around gene transcription start sites (TSSs). d Distributions of the proportion of Tn5 integration sites within peaks per nucleus. e Distribution of unique Tn5 integration sites per nucleus. f Number of nuclei that passed quality control thresholds. g–i Scatterplot displaying the number of reads per cell classified as B73 or Mo17, color-coded by genotype classification. g 16 k input B73/Mo17 scATAC-seq; h 100 k scifi-ATAC-seq; i 200 k scifi-ATAC-seq. Median contamination rate: the median cross-contamination rate, attributed to index hopping, among all predicted singlets. j UMAP of all nuclei (n = 98,424). Nuclei are colored by their predicted cell type. k Pseudobulk cell type Tn5 integration site coverage around the phloem precursor marker ZmSMXL3. Vas. par. precursor: Vascular parenchyma precursor. l Pseudobulk cell type Tn5 integration site coverage for and UMAP embeddings overlaid with gene chromatin accessibility around the ZmSMXL3 gene across all datasets. N, number of phloem nuclei

Fig. 2

Multiplexing eight samples with scifi-ATAC-seq. a Well assignment showing the multiplexing of primary samples and genotypes. b Number of profiled nuclei for each genotype. Est. Collision: estimated cell barcode collisions within the same genotype. c Spearman correlation heatmap among the 8 libraries(SS, stiff stalk; NSS, non-stiff stalk; TS, tropical/subtropical; Mixed, mixed tropical-temperate). d UMAP of all nuclei (n = 124,656). Nuclei are colored by their predicted cell type. Vas. par. precursor: vascular parenchyma precursor, procambium phloem pre.: procambium phloem precursor. e UMAP of all nuclei across the eight samples. f Chromatin accessibility of cell-type-specific accessible chromatin regions (ctACRs, Z-score > 2) among all cell types. g Pseudobulk cell type Tn5 integration site coverage around the phloem precursor marker ZmSMXL3. h Pseudobulk cell type Tn5 integration site coverage and UMAP embeddings overlaid with gene chromatin accessibility around the ZmSMXL3. N, nuclei number of procambium phloem precursor. i Chromatin accessibility of genotype-specific accessible chromatin regions (gtACRs) among seven genotypes. The number of gtACRs was labeled. j Pseudobulk cell type Tn5 integration site coverage with gene chromatin accessibility around the Zm00001eb280310 across all samples in procambium phloem precursor cells