GSM6304388: CLP, scRNAseq; Mus musculus; RNA-Seq
GSM6304388
After the lung tissue was washed twice with pre-cooled RPMI 1640+0.04% BSA medium under sterile conditions, the samples was sufficiently cut into small pieces of about 0.5 mm3.Then the samples were incubated with a mixture of 2 ml of trypsin (Gibco, Cat: R001100), 1 ml of collagenase II (Biofrox, Cat:2275MG100) and 100 µl of DNase (Servicebio, Cat: 1121MG010) for 1 h on a 37 °C shaker. The digested cell suspension was filtered 1-2 times with a BD 40 μm cell sieve, and centrifuged at 300 g for 5 min at 4°C. After the pellet was resuspended with an appropriate amount of medium, an equal volume of red blood cell lysate (MACS, Cat. No. 130-094-183) was added. After mixing, let stand at 4°C for 10 min, the cell suspension was centrifuged at 300g for 5 min, and the supernatant was discarded. The pellet was washed once with medium, centrifuged at 300 g for 5 min, the supernatant was discarded, the cell pellet was resuspended with 100 μl of medium, and the cell concentration and viability were calculated by Luna cell counter.For CD45 sorting, the prepared single cell suspensions was subjected to Magnetic-activated cell sorting (MACS) using Miltenyi mouse CD45 magnetic beads according to the manufacturer's recommendations. The CD45- and CD45+ cell suspensions were mixed 1:1 for single-cell sequencing. The single-cell library was prepared following the protocol of the 10×Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1