GSM8080217: Aim27_M2CD45neg_F2CD45pos and Aim28_M2CD45neg_F2CD45pos; Mus musculus; RNA-Seq
Following euthanization and perfusion with cold PBS through the right ventricle to clear blood and circulating immune cells, C57Bl6 mouse lungs were removed and inflated with 1-2mL of protease solution containing Collagenase Type I (catalog #17100–017, 450 U/mL; Gibco), Elastase (catalog #LS002279, 4 U/mL; Worthington Biochemical Corporation), Dispase (catalog #354235, 5 U/mL; BD Biosciences), DNaseI (catalog #10104159001, 0.33 U/mL; Roche) in DMEM/F12 (#11320033, ThermoFisher Scientific). Lungs were then diced into small pieces (<2 mm2), and incubated in 1–2 mL protease solution for 25 min at 37°C with frequent gentle agitation. Protease solution was neutralized with DMEM/F12 (#11320033, ThermoFisher Scientific) + 10% fetal bovine serum (#A3840001, ThermoFisher Scientific) and the tissue was further disrupted by gentle pipetting. Following an additional wash with DMEM/F12, the tissue was incubated for 10 min at 37°C in 2 mL Trypsin-EDTA (#59429C, Millipore Sigma) + 0.325 mg DNaseI with intermittent agitation. Trypsin was neutralized with an equal volume of DMEM-F12 + 10% FBS, and dissociated by pipetting, filtered through a 40-μm strainer, centrifuge, and resuspended in DMEM + 2% BSA. After washing, cells were stained with antibodies (CD11b (clone: M1/70), CD11c (clone: N418), CD45 (clone: 30-F11), Ep-CAM (clone: G8.8), Podoplanin (clone: 8.1.1) from BioLegend) and Fixable Viability Dye (eBioscience) for 20 minutes on ice, washed, then sorted on a BD FACSAria (BD Biosciences). For droplet-based scRNA-seq, cells were sorted into an Eppendorf tube containing 50 μl of 0.4% BSA in PBS and stored on ice until proceeding to the Single Cell Platform. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.