Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes

Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage-tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics. However, reliance on transcriptional profiling limits adaptation to other single-cell assays. With CellTag-multi, we present an approach that enables direct capture of heritable random barcodes expressed as polyadenylated transcripts, in both single-cell RNA sequencing and single-cell Assay for Transposase Accessible Chromatin using sequencing assays, allowing for independent clonal tracking of transcriptional and epigenomic cell states. We validate CellTag-multi to characterize progenitor cell lineage priming during mouse hematopoiesis. Additionally, in direct reprogramming of fibroblasts to endoderm progenitors, we identify core regulatory programs underlying on-target and off-target fates. Furthermore, we reveal the transcription factor Zfp281 as a regulator of reprogramming outcome, biasing cells toward an off-target mesenchymal fate. Our results establish CellTag-multi as a lineage-tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming. Lineage tracing using both transcriptomics and chromatin accessibility provides mechanistic insights into cell fate.

Automated summary

This study presents a new method called CellTag-multi, which allows the direct capture of heritable random barcodes expressed as polyadenylated transcripts in single-cell RNA sequencing and single-cell Assay for Transposase Accessible Chromatin using sequencing assays. The CellTag-multi method enables independent clonal tracking of transcriptional and epigenomic cell states and has been validated in studying progenitor cell lineage priming and reprogramming processes. The findings demonstrate the utility of CellTag-multi in revealing gene regulatory changes and providing mechanistic insights into cell fate.