Illumina HiSeq 2000 paired end sequencing; ChIP-seq of pancreatic cancer cells derived from a genetically engineered mouse model, which harbor the inducible KrasG12D allele
ChIP-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 ChIP-seq, at timepoints of 0 and 24 hours, DNA-protein interactions were crosslinked in iKras cells using 10% formaldehyde.  The reaction was quenched with glycine and cells harvested in PBS containing protease inhibitors. Chromatin was sheared, immunoprecipitated and processed as previously described in Lomberk, et al.(70) Supernatant was cleared by centrifugation and incubated with various histone mark antibodies (S5 Table) and 30uL of washed protein G-agarose beads (Roche 11719416001) for three days with mixing at 4C.  Following DNA purification, samples underwent real time PCR at positive and negative loci and confirmed histone mark enrichment over 1% input.  Input and ChIP DNA library preparations were completed according to NuGEN protocols with the Ovation Ultralow DR Multiplex system (NuGEN) using 10ng of DNA.