title: GSM6737821: Lung, not infected, no HDM; Mus musculus; RNA-Seq
library_construction_protocol: 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 RPMI medium, 50 μg/mL liberase TM (Roche) and 100 µg/mL DNase I (Roche), before being processed with a gentleMACS dissociator (Miltenyi) and, finally incubated for 30 min at 37 °C. 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). Cell concentration was adjusted to 1.000 total cells/μL and 12.800 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 in 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).