title: GSM8490671: KCOhom_Rep5; Mus musculus; Bisulfite-Seq
library_construction_protocol: At 13 weeks of age, mice were euthanized by CO2 asphyxiation and pancreata were rapidly dissected, flash frozen in liquid nitrogen, and stored at -80C. Tumor burden (% area of pancreas comprised of cancerous lesions) was approximately 10% and 60% for KCO(het) and KCO samples, respectively. Genomic DNA (gDNA) was purified using the DNeasy Blood & Tissue Kit (Qiagen, #69504) following the manufacturer's instructions. Snap-frozen tissues were cut into small pieces (~25 mg) and digested overnight in Buffer ATL with Proteinase K and collected using the DNeasy Mini spin column. WGBS libraries were then prepared as above using the same kit and protocol. For the KCO samples, we performed target enrichment of previously described PRC2-targeted CGIs using 200 ng of each WGBS library from two KCO(het) and five KCO mice, pooled and mixed with a Twist Custom Methylation Panel specific for targeted CGIs, Universal Blockers (Twist, #100578), and Methylation Enhancer (Twist, #103557), then dried using a SpeedVac system. The dried product was resuspended with fast hybridization mix and enhancer (Twist, #100930), and hybridized at 60°C for 2 hours. Targeted sequences were captured and purified using Streptavidin Binding beads (Twist, #100983), and the final library pool was amplified using 2x Equinox Library Amplification Mix (Twist, #104108) with 11 PCR cycles based on the total length of targeted regions. The final library was purified with DNA Purification Beads (Twist, #100983) and sequenced on a NovaSeq platform (150 bp paired-end reads).