Computational protocol: Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice

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Protocol publication

[…] Gene of interest mRNA levels were determined by real-time qPCR. Total RNA was extracted with TRIzol reagent (Ambion). qPCR was performed in triplicate using an ABI 7500 with SYBR Green PCR kits (Applied Biosystems). mRNA levels were normalized to housekeeping gene β-actin expression. All qPCR primers used are listed in .RNA-Seq reads were aligned to the mouse genome reference sequence (GRCm38/mm10) using TopHat () with a tolerance of two mismatches. Cuffdiff () was used to detect the differentially expressed genes with a cutoff of p < 0.05 and false discovery rate (FDR) < 0.25. The identified differentially expressed genes were used for pathway enrichment analysis and functional annotation with the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resources (). Heatmaps were generated in R using the gplot package. GSEA () was performed in MSigDB to generate the list of the most differentially expressed genes, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway signatures and stem cell signatures. Enriched gene sets were selected using a cutoff of p < 0.05 and FDR < 0.25 (). […]

Pipeline specifications

Software tools TopHat, Cufflinks, DAVID, GSEA
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus
Diseases Alveolar Bone Loss