GSM4497108: Low Ngr1 Rep2; Mus musculus; RNA-Seq
The small intestinal tube was dissected out and flushed with PBS to remove faeces. Small intestinal tissues were opened longitudinally, scraped with a glass coverslip to remove villi, cut into 5-mm pieces and washed with PBS five times to remove unattached epithelial fragments, mucus and faeces. Following incubation for 30 min at 4°C in 4mM EDTA-PBS solution, intestinal crypts were released from small intestinal tissue fragments by mechanically pipetting them with a 10 mL pipette in PBS and repeating this step three times. Isolated intestinal crypts were strained (70 μm cell strainer, BD Biosciences) and centrifugated three times at 1500 rpm for 2 min at 4°C. The collected crypts were maintained for 30 min at 4°C in DMEM/F12 – 10% serum (Gibco) and were then dissociated in TrypLE Express (Invitrogen) supplemented with 10 μM Rock inhibitor (Y-27632, Abcam) and DNAse 1 (Sigma-Aldrich) for 5 min at 37°C. Cell clumps and mucus were removed using a 70 μm cell strainer (BD Biosciences) and the remaining dissociated cells were washed twice with PBS collected by centrifugation at 4°C at 1500 rpm for 3 min. Antibody labelling step as well as the final resuspension of the sample were performed with PBS supplemented with 2mM EDTA, 2% FBS and 10 μM Rock inhibitor (Y-27632). Cellularized crypts were incubated with anti-CD31-BV510 (1:200, clone: MEC 13.3, BD biosciences, #563089), anti-CD45-BV510 (1:200, clone: 30-F11, BD biosciences, #563891) and anti-CD24-PeCy7 (1:100, clone: M1/69, eBioscience, #25-0242-82) antibodies in a 500µl volume for 15 min on ice. After washing twice with PBS, the cells from one animal were resuspended in a final volume of 1 mL, passed through a 70 μm strainer and transferred into appropriate FACS tubes and propidium iodide (PI) added to a concentration of 2 μg/mL. Cell sorting was carried out with a 100 μm nozzle on an Influx instrument (BD Biosciences). Aggregates, debris, dead cells (PI+) and CD45+/CD31+ hematopoietic/endothelial contaminates were depleted. For the Lgr5-GFPhigh cell population, around 2% of the CD24low Lgr5-GFP brightest cells were selected. The subsequent 2% of the CD24low Lgr5-GFP+ cells were considered as Lgr5-GFPmed and Lgr5-GFPlow cell populations. Purity of collected fractions was confirmed by reanalysis of a small fraction of the sorted cells. For single cell applications, cells were double sorted. Libraries of each biological replicate were prepared with 15ng of RNA using the Nugen Ovation RNA-Seq system V2 kit followed by Nugen Ovation Ultralow System V2 kit. High throughput sequencing was performed with HiSeq3000 with ~30 M reads targeted reads (single end 76nt length) per sample.
GEO Accession
GSM4497108