Single-cell RNA sequencing analysis of lung tissue from Tubb3-/- mice in a bleomycin-induced pulmonary fibrosis model

Whole lung cells and mural cell-enriched mesenchymal cells were isolated by cell sorting from Tubb3-/- mice and their wild-type (WT) littermates 14 days after bleomycin administration, followed by single-cell RNA sequencing analysis. Overall design: Tubb3-/- mice and their wild-type (WT) littermates were treated with bleomycin, and lung tissues were harvested 14 days post-treatment. Two biological replicates were prepared for each genotype. The isolated lungs were dissociated and processed to obtain both whole cell samples and mural cell-enriched mesenchymal cell samples. For whole cell samples, erythrocytes were removed by magnetic negative selection using rat anti-mouse TER-119 antibody followed by sheep anti-rat Dynabeads. For mesenchymal cell samples, cells were enriched by magnetic negative selection using rat anti-mouse antibodies against CD31, CD45, EpCAM, and TER-19, followed by sheep anti-rat Dynabeads. Following negative selection, both cell samples were counted and labeled with oligonucleotide tags for multiplexing using the 10x Genomics 3' CellPlex Kit Set A (1000261). The labeled samples from all four mice were pooled according to sample type (whole cell or mesenchymal cell) and stained with DAPI (0.1 µg/mL) prior to cell sorting. For the pooled whole cell sample, live cells were sorted. For the pooled mesenchymal cell sample, MCAM+ PDGFRa- (mural cells) and MCAM- (non-mural cells) populations were sorted and mixed at a 1:1 ratio to achieve mural cell enrichment. Single-cell isolation, RNA capture, cDNA preparation, and library preparation were performed according to the manufacturer's protocol (Chromium Next GEM Single Cell 3' Reagent Kit, v3.1 chemistry, 10x Genomics). Libraries were sequenced on an Illumina NovaSeq 6000 S4 flow cell. Please note that processed data files were generated from both GEX and CMO raw data, and are linked to the corresponding GEX sample records.