Bulk 3' RNA-seq Library Prep

Library preparation for bulk 3′-sequencing of poly(A)-RNA was done as described previously^{67 (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. The library was sequenced on a NextSeq. 500 (Illumina) with 16 cycles for the barcodes and UMIs in read1 and 65 cycles for the cDNA in read2.