title: Illumina HiSeq 2500 paired end sequencing; ATAC-seq of pancreatic cancer cells derived from a genetically engineered mouse model, which harbor the inducible KrasG12D allele
design: ATAC-seq of pancreatic cancer cells derived from a genetically engineered mouse model, which harbor the inducible KrasG12D allele
library_construction_protocol: iKras-p53* cell line 4292 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 ATAC-seq, iKras-p53* 4292 pellets of 5x103 cells were collected at 0 and 24h ours and resuspended in ATAC buffer and processed as described by Volk et al (A CHAF1B-Dependent Molecular Switch in Hematopoiesis and Leukemia Pathogenesis. 2018. Cancer cell 34, 707-723.e707). Briefly, the genome was fragmented by tagmentation, DNA fragments purified, and sequencing and indexing primers added by PCR.   Additional cycles of amplification, ranging from 10-14 cycles, was completed to minimally increase library content.  Libraries were recovered with the MinElute PCR Cleanup kit (Qiagen, 28004) and size selected for fragment sizes of 100-500 bp with AMPure XP beads (Beckman Coulter, A63881).