Single-cell RNA-sequencing of Organoids

Approximately five confluent organoid wells were used to harvest RNA for RNA‐sequencing (RNAseq). Organoids (ID188 passage 7, ID211 passage 6) were processed as abovementioned, and the cell pellet was resuspended in RLT buffer supplemented with β‐mercaptoethanol (Parekh et al, 2016 (link)). Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher Scientific) using oligo‐dT primer containing barcodes, unique molecular identifiers (UMIs), and an adaptor. Ends of the cDNAs were extended by a template switch oligo (TSO), and full‐length cDNA was amplified with primers binding to the TSO‐site and the adaptor. The NEB UltraII FS kit was used to fragment cDNA. After end repair and A‐tailing a TruSeq (Parekh et al, 2016 (link)), the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve better cluster recognition. The library was sequenced on a NextSeq (Macosko et al, 2015 (link)). Reference genome (GRCh38) was used for alignment. Transcript and gene definitions were used according to the GENCODE Version M25.