Abstract Single-cell genomics assays have emerged as a dominant platform for interrogating complex biological systems. Methods to capture various properties at the single-cell level typically suffer a tradeoff between cell count and information content, which is defined by the number of unique and usable reads acquired per cell. We and others have described workflows that utilize single-cell combinatorial indexing (sci) 1 , leveraging transposase-based library construction 2 to assess a variety of genomic properties in high throughput; however, these techniques often produce sparse coverage for the property of interest. Here, we describe a novel adaptor-switching strategy, ‘s3’, capable of producing one-to-two order-of-magnitude improvements in usable reads obtained per cell for chromatin accessibility (s3-ATAC), whole genome sequencing (s3-WGS), and whole genome plus chromatin conformation (s3-GCC), while retaining the same high-throughput capabilities of predecessor ‘sci’ technologies. We apply s3 to produce high-coverage single-cell ATAC-seq profiles of mouse brain and human cortex tissue; and whole genome and chromatin contact maps for two low-passage patient-derived cell lines from a primary pancreatic tumor.

High-content single-cell combinatorial indexing