Hybrid selection bisulfite sequencing of human GIST sample KIT2: Sample KIT2_HSBS
"Hybrid selection probes were designed to capture ~160,000 CTCF-binding sites, and ~5,000 promoters. CTCF bind sites lists were collated from ENCODE (as downloaded from UCSC genome browser, table wgEncodeRegTfbsClusteredV3, Release 4) as well as additional CTCF maps of primary cholangiocarcinoma and glioma16. Total genomic DNA was isolated using the DNAeasy Blood & Tissue Kit (Qiagen) and sheared using the Covaris LE220. Ampure XP beads (Agencourt) were used to size select gDNA fragments within 150-320 bp and sheared distribution was verified via BioAnalyzer (Agilient). One microgram of gDNA was end repaired, 3' A base tailed (KAPA Hyper Prep Kit no. KK8502) and ligated to sequencing adaptor (Roche SeqCap Epi Enrichment System). Ligated products were purified using Ampure XP beads. Following bead clean-up, products were bisulfite-converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) and then PCR amplified using KAPA HiFi U+ HotStart ReadyMix (KAPA no. KK2800). Equal concentrations of each library were then combined in sets of three or four along with SeqCap Epi universal and indexing oligos and bisulfite capture enhancer (SeqCap Epi Accessory Kit). Each pool was lyophilized using TOMY Micro-Vac (MV100), resuspended in hybridization buffer (SeqCap Epi Hybridization and Wash Kit), and then hybridized to SeqCap Epi Probe Pool (Roche) for 72 h at 47o C in a thermocycler. Following the 72 h incubation, captured bisulfite-converted libraries were recovered (SeqCap Pure Capture Bead Kit) at 47o C in a thermocycler for 45 min, with intermediate vortexing. Capture beads were washed (SeqCap Hybridization and Wash Kit) in a 47o C water bath. Captured bisulfite-converted libraries were then amplified via PCR (SeqCap Epi Accessory Kit). Libraries were sequenced with 10% PhiX spike-in as 100-bp end reads on the HiSeq2500 in rapid run mode."