GSM6235733: Lung, Mock, WT; Mus musculus; RNA-Seq
After euthanasia, the trachea was catheterized and BAL was performed by two consecutive flushes of the lungs with 1 mL of ice-cold PBS containing cOmplete™ Protease Inhibitor Cocktail (Roche). One tablet of inhibitor cocktail was dissolved in 50 mL ice-cold PBS. Cell density in BALF was evaluated using a hemocytometer after Tuerk solution staining (Sigma-Aldrich). To harvest lung cells, mice were perfused with ice-cold PBS through the right ventricle. Then, lung lobes were collected into a C-Tube (Miltenyi) containing complete Hanks' Balanced Salt Solution (HBSS) medium, 1 mg/ml collagenase D (Roche) and 50 μg/ml DNase I (Roche). These lung lobes were processed with a gentleMACS dissociator (Miltenyi) and, finally, were incubated for 30 min at 37 °C. Cells were finally strained through a 70-μm filter. Cell density was evaluated using a hemocytometer after Trypan solution staining (Sigma-Aldrich). Libraries preparations for single-cell immune profiling, sequencing, and post-processing of the raw data were performed at the GIGA-Genomics Core Facility (Belgium). Sorted cells were washed with 1X PBS (calcium and magnesium free) containing 0,04% weight/volume BSA (400 μg/mL). BALF and lung cells were pooled together within each condition after cell hashing with barcoded antibodies (BioLegend).We equalized numbers of cells between conditions for the scRNA-seq profiling and cell concentration was adjusted to 1.000 total cells/μL and 16.000 cells were loaded on Chromium Controller (10x Genomics). Samples were further processed for droplet-based RNA sequencing and libraries were prepared using Chromium Single Cell 3' Reagent Kits v3 according to manufacturer's protocol (10x Genomics). Amplified cDNA quality controls were performed with an Agilent bioanalyzer (Agilent) and final library profile were checked on Qiaxcel (Qiagen). Sequencing libraries were loaded an Illumina Novaseq sequencer with NovaSeq SP 100 v1 kit (Illumina, CA, USA) using the following read lengths: 28 bp for Read1 (18 bp Barcode + 10 bp Randomer), 8 bp for Sample Index and 88 bp for Read2. Library quantification was processed with KAPA Library quantification kit (KAPA Biosystems). Gene counts were obtained by aligning reads to the mm10 genome using CellRanger software (10× Genomics).