Illumina HiSeq 2000 paired end sequencing; RNA-seq of pancreatic cancer cells derived from a genetically engineered mouse model, which harbor the inducible KrasG12D allele
RNA-seq of pancreatic cancer cells derived from a genetically engineered mouse model, which harbor the inducible KrasG12D allele
iKras-p53* cell line 4292 (https://doi.org/10.1371/journal.pone.0049707) was maintained in complete media (RPMI, Gibco 11875 with 10% fetal bovine serum and 1ug/mL doxycycline) to continually express constitutively active KrasG12D.  To initiate the time course, cells were counted and split to 5E5 cells per 100mm dish and allowed to attach overnight.  The following morning, 0 hour was collected per RNA/DNA/ChIP protocols and doxycycline (1ug/mL) was added to media and allowed to incubate for 12, 24, and 48 hours prior to harvest.   For RNA-seq, at each timepoint (0, 12, 24, and 48hours), cells were washed once in PBS and harvested with RLT+ME as per RNeasy Mini Kit (Qiagen, 74106). RNA isolation included an on-column DNA digestion step and yielded >1ug per sample.  Three independent biological replicates were collected and provided to the Mayo Clinic Medical Genomics Core for sequencing.  Illumina TruSeq RNA v2 kits were used to prepare RNA libraries.