GSM3477499: WT_villi_scRNAseq; Mus musculus; RNA-Seq
Freshly harvested duodenum was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS at 4 °C. The tissue was then vigorously shaken to release the epithelium from underlying muscular tissue, and the supernatant was collected as the whole epithelium fraction; villi were collected from the top of a 70-μm cell strainer while crypts passed through. Cells were pelleted by centrifugation at 170 g at 4 °C and then washed by cold PBS. To dissociate single cells for single cell RNA-seq, isolated duodenal villi were further rotated with 5U/ml dispase (Stem Cell, #07913) and 200 U/ml of DNase I (Sigma D4513) at 37 °C for 30 min, and then washed by PBS with 1% BSA for twice, and filtered by 40-μm cell strainer. Each sample of dissociated duodenal villi was incubated with Dead Cell Removal MicroBeads (Miltenyi, USA) and passed through a magnetic column, removing the majority of dead cells/debris. The remaining live cells were counted using a trypan blue exclusion count assay with a hemocytometer and diluted for loading onto the Chromium Controller. Loading was performed to target capture of ~3,000 gel beads per sample for downstream analysis, and samples were processed through the Chromium Controller following the standard manufacturer's specifications. 10X Genomics Chromium Single Cell 3' v2 kit
GEO Accession
GSM3477499