Determination of differentiated expression states occurs after seeding of hiPS cells for cardiac directed differentiation

Single cell RNA sequencing (10X Genomics single-cell RNA-seq kit v3) of barcoded hiPS cell-derived cardiomyocytes and hiPS cells. hiPS cells were lentivirally transduced with barcode libraries consisting of random 100-mers in the 3’ untranslated region of a green fluorescent protein (GFP) transgene after which they were permitted to divide approximately three times before being split and seeded for two parallel differentiations. On day 14 of differentiation (16 days after seeding), we used fluorescent activated cell sorting (FACS) gated for GFP positivity to enrich for barcode-containing cells on which we performed single cell RNA sequencing. As transcripts of the GFP transgene are captured by single cell RNA sequencing, we were also able to recover barcodes through a separate “side reaction” cDNA sequencing run and connect them to individual cells through analysis of GFP transcript 10x sequencing reads. Separately, we also transduced hiPS cells, allowed them to divide approximately six times before harvesting them for GFP sorting and sequencing. See Jiang et al. citation for additional details and GEO accession GSE198729 for single cell RNA sequencing raw/processed data (also available on Dropbox).