RNA-Seq of explanted murine pancreatic acinar cells from wt, KrasG12D, Pi3kCAH1047R and Mek1dd mice
RNA-Seq of explanted murine pancreatic acinar cells from wt, KrasG12D, Pi3kCAH1047R and Mek1dd mice
Explanted acinar cells were collected directly after isolation (t=0 days) or after 2-3 days embedded in collagen. Collagen embedded samples were digested with collagenase for 10 min before RNA extraction. Maxwell 16 Tissue LEV Total RNA Purification Kit was used for RNA extraction according to the manufacturer's instructions (Promega) Library preparation for bulk 3'-sequencing of poly(A)-RNA was done as described previously (Parekh et al., 2016) 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.