GSM9083570: Colon tumor from Fabp (Cre/+); Apc (fl/+) mouse; Kras (LSL-G12C/+) replicate 1; Mus musculus; RNA-Seq
Dissection of mice was performed immediately after cervical dislocation. Once euthanized, mice were laid on their back and their torsos were coated with 70% Ethanol. The skin was subsequently raised, a single transverse cut of ~1.2 cm was made, and the skin was pulled in opposite directions along the coronal plane. Next, the peritoneum was carefully cut along the sagittal and transverse planes and the lower gastrointestinal track was carefully placed to one side. A cut was made through the pelvic floor adjacent to the colorectum, and the entire length of the colon, from rectum to cecum, was excised and placed in ice cold PBS. Excised colon tissue was flushed with ice cold PBS to clear fecal contents, placed on bibulous paper, and cut longitudinally. For colonic epithelium, epithelial tissue was gently scraped off the lamina propria using a razor blade and flash frozen in liquid nitrogen. For colonic tumors, macroscopic tumors were then resected and flash froze in liquid nitrogen for downstream molecular characterizations. Prior to preparation of colon and tumor tissue for western blot, RNA, and DNA analysis, tissue was pulverized under cryogenic conditions using a Bessman Tissue Pulverizer. Extraction of RNA was done using the RNeasy Plus kits from QIAGEN per manufacturer's instruction. RNA was stored at -80˚C. RNA concentration and quality was determined using an Agilent Bioanalyzer/TapeStation. 1 µg of RNA with a RIN score greater than 7 from each sample was used as input for RNA-Seq library preparation and poly-A enrichment was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module. The RNA-seq library was prepared using the NEBNext Ultra Directional RNA Library Prep kit from Illumina and quality control of the libraries was done using the TapeStation and single read sequencing of the libraries was done on a NextSeq 500 Illumina platform in a High Output mode for 75 cycles.