title: GSM5291229: Organoids from proximal part of small intestine of male old mice (105 weeks old); Mus musculus; RNA-Seq
library_construction_protocol: Small intestinal crypts were isolated using the established protocol (Sato and Clevers, 2013) with some modifications. Briefly, mouse small intestine was dissected, washed in cold PBS. The villi-free intestinal pieces (2 cm) were washed with 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 450g for 5 min at 4°C. Isolated crypts were immediately used or 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.  Crypts were resuspended in ice-cold Matrigel (Corning, Cat. 356231) and allowed to solidify at 37°C, then were supplied with culture medium (750ng/ml recombinant human Rspondin-1 (lab-made), 100ng/ml recombinant murine Noggin (PeproTech, Cat. 250-38), 50ng/ml recombinant murine EGF (PeproTech, Cat. 315-09), 10 μM CHIR99021 (Tocris, Cat. 4423/10), 100ng/ml recombinant murine Wnt-3a (PeproTech, Cat. 315-20), N2 (Thermo Fisher Scientific, Cat. 15410294), B27 (Thermo Fisher Scientific, Cat. 11500446), 1X Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. 15140122) in Advanced DMEM/F12 (ThermoFisherScientific, Cat. 11550446)). Organoids were cultured for at least 2 weeks, passaged, then IFNgamma (2ng/ml) were added at day 3 and 24 hours later being collected for scRNA sequencing. For single cell RNA sequencing: The 10X Single Cell 3' v2 reagent kit (10X Genomics) was used for cell partitioning, cDNA amplification and library construction.