title: NextSeq 500 sequencing
design: 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.
library_construction_protocol: 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.