Library preparation for bulk 3′-sequencing of poly(A)-RNA was done as described previously57 (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 after pooling of all samples full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was tagmented with the Nextera XT kit (Illumina) and 3′-end-fragments finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.57 (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 65 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.
Data were processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the ENSEMBL annotation release 75.
Data were processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the ENSEMBL annotation release 75.
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