Bulk RNA-seq Library Preparation with UMIs

Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously^{78 (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. 5’-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.^{78 (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 59 cycles for the cDNA in read1 and 18 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^{79 (link)}. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the GENCODE version M25. Differential expression analysis was performed using R and DESeq2 (1.38.0). A P value of <0.05 was used to determine differentially expressed genes. The data