NextSeq 500 sequencing; RNA-seq of mouse cell lines isolated from genetically engineered mouse models of extrahepatic cholangiocarcinoma and pancreatic cancer
RNA-seq of mouse cell lines isolated from genetically engineered mouse models of extrahepatic cholangiocarcinoma and pancreatic cancer
After 48 hours of drug treatment, when the primary cell cultures were 80% confluent, the cells were immediately transferred into RLT buffer (Qiagen) containing β-mercaptoethanol and the RNA was isolated according to the Qiagen, RNeasy kit, instructions.  Drug treated cells were seeded in a 10 cm dish, the following day the cells were treated with GDC0941, 1 micromolar, or vehicle for 48 hours. The medium containing either the drug or the vehicle was changed after 24 hours. RNA was isolated with the RNeasy kit (Qiagen).  Library preparation for bulk-sequencing of poly(A)-RNA 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.