NextSeq 500 sequencing
Libraries were sequenced on a NextSeq 500 (Illumina) with 65 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.
Barcoded cDNA for each sample was produced with Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. The ends of the cDNAs were extended with a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. UltraII FS kit (NEB) was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3â€™-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. [9], the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2, which results in better cluster recognition.