GSM5198084: young_female_organoids_inter_replication2; Mus musculus; RNA-Seq
Small intestine crypts were isolated using the established protocol (Chazaud, B. & Mouchiroud, G. Inflamm-aging: STAT3 Signaling Pushes Muscle Stem Cells off Balance. Cell Stem Cell 15, 401–402 (2014).) with minor modifications. Briefly, 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 5mM 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. For RNA isolation, the crypts were immediately resuspended in QIAzol Lysis Reagent (Qiagen) and stored at -80°C. PolyA amplification was applied.  In brief, 50 to 500 ng of total RNA were depleted of ribosomal RNA using the Ribo-ZeroTM Gold Kit H/M/R Kit (illumina) following manufacturer's instructions. Ribo-depleted RNA was resuspended in 17 μl of EFP buffer (illumina), heated to 94°C for 8 min, and used as input for first strand synthesis, using the TruSeqTM RNA Library Preparation Kit v2 (illumina) following manufacturer's instructions.