Computational protocol: Single-Cell RNA-Seq of Bone Marrow-Derived Mesenchymal Stem Cells Reveals Unique Profiles of Lineage Priming

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Protocol publication

[…] Gene expression analysis was performed with Galaxy software (Minnesota Supercomputing Institute (MSI), University of Minnesota, Minneapolis, MN). Reads were processed and aligned to the mouse reference genome (mm10_genes_2012_05_23.gtf and canonical_mm10.fa) using Tophat (version 2.0.12, open source software, http://ccb.jhu.edu/software/tophat/index.shtml)[]. See for information about the total number of reads and percent concordant mapped reads for each cell. The default options supplied with the software were used and the aligned read files produced by Tophat were processed using Cufflinks software (version 2.2.1, open source software, http://cole-trapnell-lab.github.io/cufflinks/), for further analysis, including assembling transcripts, estimating their abundance, and testing for the differential expression between single-cell RNA-seq samples[]. Read counts were normalized to fragments per kilobase of exon per million mapped reads (FPKM) according to the gene length and total mapped reads. Genes with a log2 fold change greater than 1 from the control cells to the fusion products and had a P value of less than 0.05 were considered “differentially expressed” and further analyzed for gene ontology (). Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed with DAVID informatics resources 6.7 of the National Institute of Allergy and Infectious Diseases (NIAID) and of the National Institutes of Health (NIH)()[, ]. […]

Pipeline specifications

Software tools Galaxy, TopHat, Cufflinks, DAVID
Databases KEGG PATHWAY
Application scRNA-seq analysis
Organisms Mus musculus