Computational protocol: Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro

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Protocol publication

[…] EGFP+ cells freshly isolated from mouse lung (D0) or from the colonies cultured at days 4 (D4) and 7 (D7) were used to extract RNA, 1~5 ng of total RNA was used to amplify transcripts using REPLI-g WTA Single Cell Kit (Qiagen). The amplification products were sequenced using NGS Ilumina Hiseq 2000 sequencing system (AITbiotech). Paired sequences were aligned with the mouse genome (version mm10) using Tophat2. Raw counts of each genes of each sample were calculated by HTseq. Differentially expressed genes between samples of D4 against D0, D7 against D0 and D7 against D4 were performed using the program edgeR at P-value < 0.05. The gene expression level across different samples was normalized and quantified using the function of cpm. Differentially expressed genes were annotated using online functional enrichment analysis tool DAVID ( The enrichment analysis was performed separately for different groups of genes. We compared the differentially expressed genes with those published in the single cell RNA-seq data of fetal lung cells (E18). The fetal lung single cell data included expression profiles of bipotential progenitor cells (BP), AT1s, AT2s and club cells. The single cell data were normalized for each gene with mean equal to zero across all samples. To classify the samples of our in vitro cultured Club cells (D0, D4 and D7), the expression profiles were also normalized for each gene with mean equal to zero. Expressed genes from both data sets were identified if they are not zero in at least one single cell or one sample. Expression profiles of matched genes of two datasets were hierarchically clustered with uncentered Pearson method in MeV. Both differentially expressed genes and signature genes of four cell types identified by principle component analysis (PCA) (Treutlein et al. 2014) were further analyzed to show the differentiating status of Club cells. All heatmap figures were visualized with MeV. […]

Pipeline specifications

Software tools TopHat, HTSeq, edgeR, DAVID
Application RNA-seq analysis
Organisms Mus musculus