GSM7822807: RRBS_ApcMin_Rep2_Ileum_tumor4; Mus musculus; Bisulfite-Seq
The full intestine was dissected, cleared of waste, and divided into three (Duodenum, Jejunum, Ileum) and two (Cecum and Colon) fractions for the small and large intestine, respectively. Samples were then bisected longitudinally to confirm the presence of macroscopic adenomas in the range of ~1-5 mm, after which the tumors were carefully dissected and the position from the cecum and diameter of the tumor were measured. Following isolation of visible benign tumors, the bulk epithelial tissue was purified to provide a matched non-cancer tissue control. Both primary “tumors” and isolated epithelia were briefly inspected for purity prior to snap freezing on dry ice. Then, isolated tissue were purified using QIAmp DNA Blood and Tissue Kit with an extended 6 hour Proteinase K incubation to improve the bisulfite conversion efficiency. Concentration of genomic DNA (gDNA) was quantified using a Qubit 3.0 Fluorometer. RRBS was performed on 100 ng gDNA of each sample using the Swift Biosciences ACCEL-NGS Methyl-Seq DNA Library Kit (#30024) following the manufacturer's recommendations with the following modifications: The DNA was fragmented using Covaris S2 and purified with Agencourt RNAclean XP beads (Beckman Coulter, #A63987) (1.8X). After the final repair step, the bisulfite conversion of DNA was conducted using the Qiagen EpiTect Fast Bisulfite Conversion kit (Qiagen, #59824) following the manufacturer's recommendations, eluting the bisulfite converted DNA in 22 µl EB. Libraries were amplified with 10 cycles of PCR. Amplified library purification with Agencourt RNAclean XP beads (Beckman Coulter, #A63987) was performed twice (1.2X). The purified libraries were sequenced for 76 bp single-end reads on a NextSeq 500  platform (Illumina).