title: GSM9057711: Lungs of Gprc5a-/- mice exposed to anti-IL-1𝛽 and anti-PD-1 and analyzed at 3 months post-NNK, replicate 1; Mus musculus; RNA-Seq
library_name: GSM9057711
library_construction_protocol: scRNA-seq experiment n = 31 mice: Lungs were obtained from Gprc5a−/− exposed to NNK and one of four antibody treatments. At each defined end point, lungs were processed into single cells by enzymatic digestion in a solution composed of collagenase type I (CLS-1 LS004197, Worthington), elastase (ESL LS002294, Worthington) and DNase I (D LS002007, Worthington). Samples were transferred to 5 ml LoBind Eppendorf tubes and incubated in a 37 °C oven for 20 min with gentle rotation. Samples were then filtered through 70 μm strainers (Miltenyi Biotech, 130-098-462) and washed with ice-cold HBSS. Filtrates were then centrifuged and resuspended in ice-cold ACK lysis buffer (A1049201, Thermo Fisher Scientific) for red blood cell lysis. Following red blood cell lysis, samples were centrifuged and resuspended in ice-cold FBS, filtered (using 40 μm FlowMi tip filters; H13680-0040, Millipore) and an aliquot was taken to count cells and check for viability by Trypan blue (T8154, Sigma Aldrich) exclusion analysis. Cell suspensions were then sorted to enrich for epithelial cells. Namely, mouse lung single cells were stained with Sytox Green and the following cocktail of antibodies: CD45-PE/Cy7 (BioLegend, 103114), ICAM2-A647 (Life Technologies, A15452), EPCAM-BV421 (BioLegend, 118225) and ECAD-A488 (eBioscience, 53-3249-80). To enrich epithelial cells, live (Sytox-) epithelial singlets (CD45-; ICAM2; EpCAM+ &/or ECAD+) were sorted and mixed at a 1:1 ratio with live cells from each sample. Mixtures were then loaded on a Chromium Controller (10x Genomics, 110211) and 5′ single-cell gene expression libraries were prepared using the Chromium Next GEM Single Cell 5′ Gel Bead kit v.1.1 (10x Genomics, 1000169). cDNA