GSM5905270
The right caudal lope of the lung was removed from the body and transferred on ice in PBS containing 1% BSA (w/v) and 2 mg/ml Actinomycin D (Sigma-Aldrich) for further processing. Lung tissue was incubated in 2 mL Dispase (Corning, Bedford, MA, USA) containing 2 mg DNAse (AppliChem, Darmstadt, Germany), 4.6 mg Collagenase B (Roche, Basel, Switzerland) and 2 mg/ml Actinomycin D (Sigma-Aldrich) and at 37 °C for 30 min. Digest was stopped by the addition of cold PBS containing 0.5% (w/v) BSA and Actinomycin D. The tissue was disrupted by pipetting in a repeated pumping motion. Cell suspension was collected and filtered through 70 mm-filter to obtain a single-cell suspension. Red Blood cell were lysed by incubation with RBC lysis buffer (Santa Cruz Biotechnology, Dallas, Texas, USA) for 4 min at room-temperature. Lysis reaction as stopped by the addition of PBS containing 0.04% BSA, the supernatant was removed by centrifugation (1,200 rpm, 6 min, 4 °C) and cells were resuspended in PBS containing 0.04% BSA. Barcoding of single cells and RNA Isolation was performed using the Chromium controller and Chromium Next Gem Chip G Single Cell Kit and Chromium Next Gem Single cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics B.V., Leiden, The Netherlands) according to the manufactures instructions. After enzymatic fragmentation and size selection, resulting double-stranded cDNA amplicons optimized for library construction were subjected to adaptor ligation and sample index PCRs needed for Illumina bridge amplification and sequencing according to the manufacturer's instruction (10x genomics). Single cell libraries were quantified using Qubit (Thermo Fisher) and quality-controlled using the Bioanalyzer System (Agilent). Sequencing was performed on a Novaseq 6000 (Illumina), aiming for 200 Million reads per library (read1: 28, read2: 150 nucleotides).