Generalization of the sci-L3 method to achieve high-throughput linear amplification for replication template strand sequencing, genome conformation capture, and the joint profiling of RNA and chromatin accessibility

Single-cell combinatorial indexing (sci-) methods have addressed major limitations of throughput and cost for many single cell modalities. With the incorporation of linear amplification and 3-level barcoding in our suite of methods called sci-L3, we further addressed the limitations of uniformity in single cell genome amplification. Here, we build on the generalizability of sci-L3 by extending it to template strand sequencing (sci-L3-Strand-seq), genome conformation capture (sci-L3-Hi-C), and the joint profiling of RNA and chromatin accessibility (sci-L3-RNA/ATAC). We demonstrate the ease of adapting sci-L3 to these new modalities by only requiring a single-step modification of the original protocol. As a proof-of-principle, we show our ability to detect sister chromatid exchanges, genome compartmentalization, and cell state specific features in thousands of single cells. We anticipate sci-L3 to be compatible with additional modalities, including DNA methylation (sci-MET) and chromatin associated factors (CUT&Tag), and ultimately enable a multi-omics readout of them. We performed three extensions of the sci-L3 methodology. Profiling replication template strand in single cells using sci-L3-Strand-seq was performed on mouse diploid Patski cells and human haploid HAP1 cells. Single cell genome conformation capture was performed with the GM12878 cell line. The joint profiling of RNA and chromatin accessibility using sci-L3-RNA/ATAC was performed on HEK293T, BJ-5ta, NIH/3T3 cell lines in parallel with sci-L3-RNA/DNA.