GSM8381032: High_rep2; Mus musculus; RNA-Seq
Mouse small intestine was dissected, washed in cold PBS and opened longitudinally. Villi were removed by scraping with a coverslip. The villi-free intestinal pieces (2 cm) were washed four times in cold PBS and transferred to 5 mM EDTA/PBS, followed by two 30-min incubations at 4°C on a rotator. The tissue was transferred to fresh cold PBS and manually shaken for 30 sec. The crypt solution was filtered using a 70 µm cell strainer and centrifuged at 120xg for 5 min at 4°C. The crypts pellet was resuspended in 1 ml of cold PBS and evaluated for purity microscopically. Isolated crypts were aliquoted, snap frozen in liquid nitrogen and stored at -80°C for further experiments.  Genomic DNA and RNA from single crypts were isolated using Quick-DNA/RNA Microprep Plus Kit (Zymo Research) following the manufacturer's instructions.  We processed around 150 crypts from old mice, analyzed their DNAm level by Pyrosequencing on the 4 gene promoters (Dkk1, Dkk2, Sfrp1 and Sfrp2), calculated the average DNAm level, then we selected the crypts of the top 10% (high 5mC crypts) and the bottom 10% (low 5mC crypts) and performed single-crypt RNAseq. The RNAseq library preparation of single crypts was performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit (NEB) following the manufactures' instructions.