GSM7866907: RUN1_WT_mRNA; Mus musculus; RNA-Seq
The small intestine was collected from WT or VilCreIfnlr1fl/fl mice three days after targeted irradiation. The luminal content was removed by flushing with HBSS (without Mg2+/Ca2+). The epithelial tissue was then dissociated by incubating with HBSS containing Pen/Strep (100X), 10 mM HEPES, 2% FBS, and 15 mM EDTA in a shaking incubator. The intestinal crypt cells were isolated by passing through 100 µm and 70 µm strainers and then stained with an anti-EPCAM Biotin and an anti-CD45 FITC antibody. The cells were washed with FACS buffer (PBS, 5% BSA, 2 mM EDTA), followed by staining with TotalSeqB PE Streptavidin. After washing, the cells were resuspended in PBS, stained with a viability dye, and then washed and resuspended in FACS buffer. Live epithelial cells were sorted using a FACSAria III (Becton Dickinson) based on the presence of the PE signal and the absence of the FITC signal. The sorted cells from different samples were pooled into one WT and one VilCreIfnlr1fl/fl sample in PBS supplemented with 0.04% BSA. HTO and DNA libraries were sequenced at the CIML genomic facility using a P3 flowcell in Illumina NextSeq 2000. 2 independent experiments were performed and analyzed separately.