txci-ATAC-seq: a massive-scale single-cell technique to profile chromatin accessibility

We develop a large-scale single-cell ATAC-seq method (txci-ATAC-seq) by combining Tn5-based pre-indexing with 10X Genomics barcoding. Leveraging this molecular hashing strategy, we demonstrate that txci-ATAC-seq enables the indexing of up to 200,000 nuclei across multiple samples in a single emulsion reaction, representing a ~22-fold increase in throughput compared to the standard workflow at the same collision rate. To improve the multiplexing capability of this new technique, we further develop a "phased" protocol variant (Phased-txci-ATAC-seq) that effectively decouples sample processing from library preparation and has the potential to profile up to 96 samples simultaneously. In this study, we profile 449,953 nuclei across diverse tissues, including the human cortex, mouse brain, human lung, mouse lung, mouse liver, and lung tissue from a club cell secretory protein knockout (CC16-/-) model. Our study of CC16-/- nuclei uncovers previously underappreciated technical artifacts derived from remnant 129 mouse strain genetic material, which cause profound cell-type-specific changes in regulatory elements near many genes, thereby confounding the interpretation of this commonly referenced mouse model. We extracted nuclei from multiple sources, including GM12878 and CH12.LX cell lines, human lung tissue, native lung and liver samples from two 24-week-old male C57BL/6J mice, and lung tissue from age-matched (~8 weeks) wild-type (n=3) and CC16 deficient (n=3) mice on a C57BL/6J background. Two different barnyard settings were designed to estimate the total collisions arising from pre- and/or post-pooling events. To test the total collision rate, the human and mouse cells were mixed in the same well at a 1:1 ratio to perform barcoded transposition (“true barnyard”). The collision rate driven by events downstream of pooling was tested by performing barcoded transposition on wells containing pure species (“pseudo-barnyard”) and pooling the human and mouse nuclei afterward.