RNA-seq of intestinal epithelial stem cells that have an enrichment of either young or old mitochondria
RNA-seq of intestinal epithelial stem cells that have an enrichment of either young or old mitochondria
Mouse small intestinal crypts were isolated as follows. Small intestines were flushed with ice-cold PBS and opened longitudinally, and mucus was removed by gentle rubbing. The intestine was cut into small fragments and incubated with three changes of 10 mM EDTA in PBS on ice for 1 h and 45 mins. Epithelium was then detached by vigorous shaking. To enrich for crypts, the tissue suspension was filtered through 70-μm nylon mesh. Single cells were isolated by dissociating crypts in TrypLE Express (Gibco) with 1,000 U/ml of DNase I (Roche) at 32 °C for 2 minutes. Cells were washed and stained with the following antibodies: CD31–PerCP-Cy5.5 (BD, Mec13.3), CD45–PerCP-Cy5.5 (BD, 30-F11), Ter119– PerCP-Cy5.5 (BD Ter119), CD326–BV786 (BD, G8.8) and CD24–BV421 (BD, M1/69), all at 1:500 dilution. Finally, cells were resuspended in HBSS containing 0.2% BSA supplemented with 7-AAD (Life) (2 μg/ml) for live-cell separation. Cells were sorted using a FACSAria Fusion (BD Biosciences). RNA extraction was performed using Trizol according to manufacturer's instructions for RNA isolation. Samples were first treated with HL-dsDNase (Articzymes 80200-050) to remove residual DNA. NuGEN Ovation SoLo RNA-Seq kit was used for Illumina library preparations (NuGEN Technologies).