title: GSM8494224: lung, Smad4iko, sample 3; Mus musculus; RNA-Seq
library_construction_protocol: Mouse lung tissue at the peripheral region distal to the pulmonary hilum was collected from mice at age 8 weeks. To prepare single-cell suspension, tissue blocks were minced and digested in an enzyme-containing buffer (1 mg/ml type I collagenase; 0.5 mg/ml type IV collagenase; DNase I, 1 U/ml; 2 mM MgCl2; 2 mM CaCl2) for 50 min at 37°C with occasional shaking. Thereafter, heat-inactivated fetal bovine serum (FBS, 10% v:v) was added to neutralize the enzymes. The tissue digest was further dissociated by repeated pipetting and gentle mincing and then filtered through a 70-μm strainer and a 40-μm strainer to remove undigested cell aggregates. After lysing red blood cells, the remaining pulmonary single cells were collected, rinsed and re-suspended in 0.04% BSA/PBS, followed by Acridine Orange/Propidium Iodide staining for cell count and viability test. In each preparation, we obtained approximately 1X10^6 cells per mL and the viability was more than 80%. Library construction were performed according to manufacter's instruction for single cell 3' 10x genomic and prepared for sequencing using standard Illumina protocols. Library were sequenced using the NovaSeq System (Illumina) with a 150 bp paired-end run according to the manufacture's instruction.