GSM7295752: SpcPT, replicate 2, scRNAseq; Mus musculus; RNA-Seq
SpcT and SpcPT mice treated as described above. Due to mouse colony limitations, one SpcT mouse was Sftpc-CreERT2;p53+/+;Rosa26Confetti (Brainbow 2.1)85. However, RFP is only expressed after Cre treatment, as in SpcT mice, and only red RFP+ cells were sorted from this mouse in an identical manner as the replicate SpcT mouse. 96 hours after BHT treatment, lungs were harvested and perfused with 10 mL of ice-cold PBS and placed in a sterile dish on ice. Lungs were minced with sterile scissors and resuspended in 6 mL digestion media (RPMI with 0.083 U/mL of collagenase type IV (Sigma-Aldrich), 0.6 U/mL dispase ii (Sigma-Aldrich), and 25 µg/mL DNase (Sigma Aldrich)).  Lungs were rotated for 30 minutes at 37ºC. Tubes were briefly cooled on ice, and then samples were passed through a 70 µM filter followed by a 40 µM filter. 5 mL of FACS buffer (10% FBS, 2 mM EDTA, 25 µg/mL DNase in DPBS) was added and tubes were spun for 5 minutes at 300g. Cells were resuspended in ACK lysing buffer (Gibco), incubated for 1 minute on ice, and quenched with 8 mL of FACS buffer. Cells were washed 2x with FACS buffer, resuspended with biotinylated primary antibodies (CD45, BioLegend 103104 30-F11; CD31, BioLegend 102404 390; F4/80, BioLegend 123106 BM8; Ter119, BioLegend 116204 TER-119, 1:800 in FACS buffer) and incubated for 20 minutes on ice. After washing 2x, cells were resuspended in streptavidin-APC secondary antibody (BioLegend 405207, 1:800) for 20 minutes on ice. Cells were washed 2x and resuspended in FACS buffer with 1 µg/mL DAPI, filtered through a 40 µM filter, and sorted using a Sony SH800S cell sorter. Data were analyzed using the default Sony SH800S software and FCS Express (Version 7, De Novo Software). Single cell suspensions were prepared from mice treated with BHT as described above. Libraries were prepared using the 10x Genomics Single Cell 3' Library v3.1 kit according to the manufacturer's protocols. An input of 10,000 cells were added to each 10x channel. Libraries were sequenced on an Illumina NovaSeq6000.