title: GSM3930560: vehicle-treated colon_rep 4; Mus musculus; RNA-Seq
library_construction_protocol: Intestinal tissues were harvested from mice. About 10-15 cm of the proximal half of small intestine were used for the duodenum crypts and villi samples, and the entire colon tissues from the terminal cecum to rectum were used for colon samples. After flushing with cold PBS, the intestines were cut opened longitudinally, cut into 1 cm pieces in cold PBS for washing, and then incubated with 3mM EDTA/PBS in rotator for 5 min at 4 0C. The solution was discarded and the tissues were incubated with fresh 3mM EDTA/PBS in rotator for 10 min at 4 0C. After shaking the tubes gently for 10 times, the intestinal pieces were transferred to new tube prepared with cold 3mM EDTA/PBS, rotated for 30 min at 4 0C, then were shaken 30 times. The supernatant containing the whole epithelium tissues (crypts and villi) were collected and spun down at 200 rcf for 3 min at 4 0C. Resuspended the pellet with cold PBS and separated villi from crypts with 70 µm filter. Collected both villi and crypts in different Eppendorf tubes. Centrifuged tubes at 200 rcf for 3 mins at 4 0C and at 300 rcf for 30 seconds at 40C in order to remove any residual PBS. Proceeded to RNA extraction. For the colon samples, on an ice-cold plate, the colon was cut from the terminal end of the cecum to the rectum.   An 18-gauge needle attached to a 30 ml syringe was used to flush the feces out of the colon. The colon was cut open longitudinally and rinsed with cold PBS. Afterwards, the colon was scraped with a glass slide and a glass cover slip. The colon was scraped until the tissue turned transparent and only the muscle tissue was left. The scraped epithelial cell pellet was then collected in a 1.5 ml Eppendorf tube coated with 0.5% BSA and filled with 1ml of cold PBS. After pelleting the colon intestinal cell pellet in cold PBS at 300 rcf for 3 minutes in 4C twice, the supernatant was removed. Trizol was quickly added to the pellet by pipetting up and down until the pellet dissolved in the Trizol.RNeasy Plus Universal Kit was used for villi and crypts with RiboZol RNA extraction reagent (Amresco, Solon, Ohio), according to manufacturer's instructions. All nucleic acid extracts were gDNA Eliminator Solution for 15 s at 37 °C, in order to remove contaminating chromosomal DNA. Resulting RNA was analyzed for quantity and quality with a NanoDrop spectrophotometer ND-1000 (Isogen Life Science, Utrecht, The Netherlands). RNA quality was examined via Agilent 2100, mRNA was enriched and reverse transcribed, and DNA nanoball synthesis was conducted by BGI. Sequencing was performed on BGISEQ-500 with paired-end, 100bp reads.
attributes.tag: GEO Accession
attributes.value: GSM3930560