title: GSM9102678: NP treated lung, sorted NP+CD45+; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were explanted and mechanically minced with scalpels and enzymatically digested with TM liberase (Roche) and 150 U/mL DNase I (Sigma) for 30 minutes at 37°C. Digested lungs were then passed through 70 μm cell strainers (Corning) and washed with DPBS supplemented with 0.5% bovine serum albumin and 2mM EDTA (MACS buffer). Lungs were then processed with ACK buffer (Gibco) for 5 minutes at room temperature to lyse erythrocytes and quenched with MACS buffer. The resulting single cell suspensions were resuspended at a concentration of 1e6 cells/100 μL for flow cytometry sorting. Cells were then prepared for flow-assisted cell sorting by staining with AF488-CD45 (Biolegend) and sorted into CD45+NP+, CD45+NP-, and CD45- populations on the Bigfoot Cell Sorter (Invitrogen). Primary tumors were excised, mechanically minced with scalpels and enzymatically digested using the tumor dissociation kit (TDK, Miltenyi), incubated for 30 min at 37°C. Digested PTs were then passed through 70 μm cell strainers (Corning) and washed with DPBS supplemented with 0.5% bovine serum albumin and 2mM EDTA (MACS buffer). PTs were then processed with ACK buffer (Gibco) for 5 minutes at room temperature to lyse erythrocytes and quenched with MACS buffer. Single cell suspesions of lung and PT were treated with Chromium Fix RNA Profiling kit according to manufacturers instructions. Chromium Fix RNA Profiling (10x Genomics) using the Chromium Mouse Transcriptome Probe Set v1.0.1.