GSM3440072: SC02; Mus musculus; RNA-Seq
Mice were euthanized with sodium pentobarbital overdose. Chest cavity was opened and lungs were perfused through the right ventricle with 10 ml of HBSS. The lungs were removed and, using 30G needle, infused with 1 mL dispase (Corning) with DNase I (Sigma). Lungs were incubated at room temperature with gentle agitation for 45 minutes, followed by gentle teasing using forceps into small (1-2 mm) fragments and incubation in digestion buffer for another 15 minutes. Resulting suspension was passed through passed through 70 μm cell strainer (Falcon), washed with DMEM (Corning) supplemented with 5% FBS (Corning), pelleted by centrifugation and erythrocytes were lysed using BD Pharm Lyse (BD Biosciences). Resulting single cell suspension was kept in DMEM/FBS and passed through 40 μm cell strainer (Falcon) two times. Cells were counted using Cellometer K2 (Nexcelom) with nucleic acid binding dyes acridine orange (AO) to calculate total number of nucleated cells and propidium iodide (PI) to count dead cells. All manipulations were performed using wide bore tips (Axygen). 10x Genomics, 3' single-cell RNA-seq, V2 chemistry (Protocol CG00052 Rev B). Read 1 (26 cycles) corresponds to cell barcode and UMI, Read 2 (98 cycles) corresponds to 3'-end of RNA molecule. Index 1 (8 cycles) is single cell library barcode.
GEO Accession
GSM3440072