GSM6801674: Lung, PUUC 4 h, rep 1; Mus musculus; OTHER
Mice were euthanized at 4 and 24 h, and lungs were processed into single-cell suspensions with a gentle MACS Octo Dissociator and Mouse Lung Dissociation Kit (Miltenyi Biotec, #130-095-297) according to manufacturer protocols. Cell suspensions were counted and evaluated for viability over 80%. Live cell enrichment (e.g., Miltenyi Biotech) was processed if the cell viability was less than 80%. Suspensions were centrifuged at 400 x g for 5 min and were washed immediately after diluting the samples in 10ml PBS + 0.1% BSA. Cells were counted using Nucleocounter (Cemometec) automated counters. Cells were blocked with 0.5 µL of TruStain FcX™ PLUS (anti-mouse CD16/32) antibody and were incubated at 4°C for 10 min. The antibody cocktail was added to stain the cells and incubated for 30 minutes before proceeding to the wash steps. Three wash steps were performed, followed by filtration. Following cell viability confirmation, the 10x Genomics single cell 3' v3.1 assay protocol was performed according to the manufacturer's instructions (transcriptome: mm10-2020-A). To obtain sufficient read coverage for both libraries, we sequenced ADT libraries in 10% and cDNA library fraction at 90% of a lane (Novaseq6000 SP kit).