NextSeq 500 sequencing; RNA-seq of pancreatic mouse tumor cell lines
RNA-seq of pancreatic mouse tumor cell lines
Tumors from Ptf1aCre/+;KrasG12D/+ or Pdx1-Cre;KrasG12D/+ mice were removed and digested in 10 mL Dulbecco's modified Eagle medium (DMEM) containing 150 U/mL collagenase Type 2 (Worthington, Lakewood, NJ) as previously described. Digested tumors were dispersed into single cell suspension, resuspended in DMEM medium containing 10% fetal calf serum (FCS), and cultivated as described previously. All human cell lines were obtained from ATCC except the primary dispersed human PDAC primary cell lines, Loh-C and SMJ31, which were obtained from pancreatic ductal adenocarcinoma resections. After 24 hours of drug treatment, when the primary cell cultures were 80% confluent, the cells were immediately transferred into RLT buffer (Qiagen) containing -mercaptoethanol and the RNA was isolated according to the Qiagen, RNeasy kit, instructions.  For drug treated cells only as indicated in the sample attributes. Cells were seeded in a 10 cm dish, the following day the cells were treated with Trametinib, 10 nanomolar, or vehicle (DMSO) for 24 hours.  RNA was isolated with the RNeasy kit (Qiagen).  Library preparation for bulk-sequencing of poly(A)-RNA by UMI-seq was done as described previously (Parekh et al., 2016). 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 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. 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. (2016), 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.