Illumina HiSeq 2000 paired end sequencing; RRBS of pancreatic cancer cells derived from a genetically engineered mouse model, which harbor the inducible KrasG12D allele
RRBS 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/ChIPprotocols and doxycycline (1ug/mL) was added to media and allowed to incubate for 12, 24, and 48 hours prior to harvest.   For RRBS-seq, at each timepoint (0, 12, 24, and 48 hours), cells were washed once in PBS, detached with a scraper in 1mL PBS, and pelleted prior to preservation at -20°C.  DNA was isolated from the cell pellets using the QIAamp DNA Mini and Blood Mini Kit (Qiagen, 51304). A single biological condition was collected and provided to the Mayo Clinic Medical Genomics Core for bisulfite conversion, library preparation and sequencing.  Briefly, DNA (250ng) was digested with Msp1 (New England Biolabs (NEB), R0106M) and purified using Qiaquick Nucleotide Removal Kit (Qiagen, 28004).  End-repair A tailing was performed (NEB, M0212L) and TruSeq methylated indexed adaptors (Illumina, 15025064) were ligated with T4 DNA ligase (NEB, M0202L). Size selection was performed with Agencourt AMPure XP beads (Beckman Coulter, A63882). Bisulfite conversion was performed using EZ-DNA Methylation Kit (Zymo Research, D5001) as recommended by the manufacturer with the exception that incubation was performed using 55 cycles of 95 ̊C for 30 sec and 50°C for 15 min.  Following bisulfite treatment, the DNA was purified as directed and amplified using Pfu Turbo C Hotstart DNA Polymerase (Agilent Technologies, 600414).   Library quantification was performed using Qubit dsDNA HS Assay Kit (Life Technologies, Q32854) and the Bioanalyzer DNA 1000 Kit (Agilent Technologies, 5067-1504). The final libraries from RRBS were prepared for sequencing as per the manufacturer's instructions in the Illumina cBot and HiSeq Paired end cluster kit v3.