GSM6995423: Lung tissue, healthy control, a 1-year boy with lung sequestration, scRNAseq; Homo sapiens; RNA-Seq
For each participant, 5mL venous blood was collected in a BD Vacutainer EDTA Tube (BD, Cat#: 367861). The samples were density gradient centrifuged to obtain peripheral blood cells using the Ficoll-Paque medium (PMID:33564978). Lung tissues were minced and digested to prepare single-cell suspension. Single-cell suspension with the concentration of 7-12X10^5 cells/mL. For single-cell suspensions of lung tissues, the cell viabilities were 56-75%. For single-cell suspensions of peripheral blood cells, cell viabilities were higher than 80%. Each single-cell suspension was used to constructing a single-cell RNA library according to 10x Genomics® Cell Preparation Guide (PMID:35724763;34521850). The single-cell RNA-seq raw data were aligned and counted against the human reference genome (GRCh38) using Cell Ranger (version 4.0.0) to obtain gene-cell matrices. After removing doublet cells with DoubletFinder (version 2.0.3), the matrices were submitted to Seurat package (version 4.1.1) to achieve quality control, batch effect removal, dimension reduction and clustering analysis. Quality control: total RNA counts between 200 and 6000, and mitochondrial gene percentage <10%. The top 2,000 most variable genes were obtained for dimensional reduction by “FindVariableFeatures”, “ScaleData” and “RunPCA” function. After batch effect removal with Harmony package (version 0.1.0), “FindNeighbors”, “FindClusters” and 'RunTSNE' were performed to identify the cell clusters. Finally, the two-dimensional profiles were visualized by t-Distributed Stochastic Neighbor Embedding (t-SNE). The cell types were identified based on canonical marker genes (PMID:35724763).