Bulk 3' transcript end RNA sequencing of mouse intestinal tissues across defined stages of Kras(G12D)-driven serrated intestinal tumor progression.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) from intestinal tissues of wild-type mice and Kras(G12D) mice representing defined stages of serrated intestinal tumor progression (hyperplasia, adenoma, and carcinoma; n = 24) and subjected to bulk 3' transcript end RNA sequencing. Barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher Scientific) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. 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 adaptor. NEBNext Ultra II 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. (Sci Rep. 2016 May 9:6:25533) 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 550 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.
The 3-prime RNA sequencing was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables (Macosko et al. [Cell. 2015 May 21;161(5):1202-1214]). Reference genomes GRCm38 and GRCh38 were used for alignment of mouse and human samples, respectively. Transcript and gene definitions were used according to the Gencode v.38 62. The data was processed in R using the DESeq2 package (v1.36) for the read normalization and variance stabilizing transformation (Love et al. [Genome Biol. 2014;15(12):550]).