Abstract
A major challenge in developmental biology is to understand the genetic and cellular processes/programs driving organ formation and differentiation of the diverse cell types that comprise the embryo. While recent studies using single cell transcriptome analysis illustrate the power to measure and understand cellular heterogeneity in complex biological systems, processing large amounts of RNA-seq data from heterogeneous cell populations creates the need for readily accessible tools for the analysis of single-cell RNA-seq (scRNA-seq) profiles. The present study presents a generally applicable analytic pipeline (SINCERA: a computational pipeline for SINgle CEll RNA-seq profiling Analysis) for processing scRNA-seq data from a whole organ or sorted cells. The pipeline supports the analysis for: 1) the distinction and identification of major cell types; 2) the identification of cell type specific gene signatures; and 3) the determination of driving forces of given cell types. We applied this pipeline to the RNA-seq analysis of single cells isolated from embryonic mouse lung at E16.5. Through the pipeline analysis, we distinguished major cell types of fetal mouse lung, including epithelial, endothelial, smooth muscle, pericyte, and fibroblast-like cell types, and identified cell type specific gene signatures, bioprocesses, and key regulators. SINCERA is implemented in R, licensed under the GNU General Public License v3, and freely available from CCHMC PBGE website, https://research.cchmc.org/pbge/sincera.html.
Highlights
Genetic and phenotypic heterogeneity among cells is a general phenomenon, associated with the development of biological function and disease processes [1,2,3]
Through the application of SINCERA to analyze RNA-seq data from single cells isolated from whole fetal mouse lung at E16.5, we demonstrated its utility and accuracy
Two independent experiments of 96 chambers single cell RNA-seq have been performed; sequence alignment to the mouse genome using Cufflinks [37]; quality controls were done in CCHMC DNA Core using standard protocols
Summary
Genetic and phenotypic heterogeneity among cells is a general phenomenon, associated with the development of biological function and disease processes [1,2,3]. The epigenetic status, cell cycle, microenvironment and intrinsic transcriptional ‘noise’ are all likely to influence the extent of heterogeneity within a seemingly homogenous cell population within an organ [4,5,6]. Cell fate decisions during organ development are largely operative at the level of individual. A Single-Cell RNA-Seq Analytic Pipeline design, data collection and analysis, decision to publish, or preparation of the manuscript
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