GSM5725943: EC-Foxc-DKO Rep 2; Mus musculus; RNA-Seq
For single-cell RNA sequencing, mouse distal jejuna were collected 18.5h after small intestinal ischemia/reperfusion surgery. Two mice were used for each group: Control I/R-18.5h and EC-Foxc-DKO I/R-18.5h. Briefly, mice were anesthetized by isoflurane. Blood was removed by cardiac perfusion with cold PBS. The distal jejunum was then dissected from each individual, washed with cold PBS and cut into small pieces. The tissue was processed for scRNA-seq upon dissociating into single cell suspension in a digestion buffer (2 mg/mL Collagenase D, 0.2mg/mL DNAase I, 2 mg/mL Dispase II, 100 units/mL penicillin and 100 µg/mL streptomycin in HBSS) for 35 min at 37°C, followed by filtration through a 70μm and a 40μm cell strainer. Cells were washed with washing buffer (0.5% BSA, 2mM EDTA in PBS, pH 7.4) for three times, and resuspended in PBS with 0.04% BSA at a concentration of 1200 cells/μL (according to 10x Genomics Document #CG00053 Rev B) before being passed through a 30 μm MACS SmartStrainer. The cell viability was tested by using the Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, USA). The cell sample was processed for scRNA-seq only when the cell viability was more than 70%. Single cell 3' gene expression libraries were constructed from samples by using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics, Pleasanton, CA, USA) according to the manufacturer's manual CG000204 Rev D. The single cell libraries were then assessed for quality (TapeStation 4200, Agilent, Santa Clara, CA, USA) and then run by using paired-end 50 bp sequencing on the Illumina HiSeq 4000 platform (Illumina, San Diego, CA, USA). 10,000 cells were targeted for each sample with a sequencing depth of 20,000 read pairs per cell.