Poly(A)-RNA Bulk Sequencing Protocol

Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously^{36 (link)}. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 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 adapter. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing, a TruSeq adapter was ligated and 3'-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.^{36 (link)}, the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables. Reference genome (GRCm38) was used for alignment^{37 (link)}. Transcript and gene definitions were used according to the GENCODE Version M25. Heatmaps shown display the log2 fold change.