title: GSM3477499: WT_villi_scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: 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
attributes.tag: GEO Accession
attributes.value: GSM3477499