title: GSM6720373: Room Air; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were perfused with PBS, minced with scissors and digested in an enzymatic mixture (dispase II: 200 μg/ml, collagenase I: 200 μg/ml, DNase I: 40 μg/ml) for 35 min at 37°C. Following enzymatic digestion, the samples were filtered, and red blood cells were lysed. Then, single cells were resuspended in MACS buffer (DPBS + 0.1% BSA + 2 mM EDTA). Ensure cell viability exceeded 80% as determined by AO/PI reagent staining. The following sequencing steps were performed by 10X Genomics. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software (version 2.20). Cell Ranger pipelin (version 6.1.1) was used for sample demultiplexing, barcode processing and single-cell 3' gene counting, and scRNA-seq data were aligned to the reference genome. Seurat (Version 4.0) was used for dimensional reduction, clustering and analysis of scRNA-seq data. Each group contained eight mice mixed into one sample for scRNA-seq. Library was performed according to the manufacturer'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 Illumina 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 appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.