Computational protocol: A multi-step transcriptional cascade underlies vascular regeneration in vivo

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Protocol publication

[…] Library preparation was performed using the SMARTer Stranded Total RNASeq Kit Pico Input (Takara Bio USA, Mountain View, CA) according to manufacturer’s instructions. Sequencing was performed on a HiSeq. 3000 instrument (Illumina, San Diego, CA) using parameters optimized for 50 bp single-end reads at a depth of 20 million reads/sample. Raw sequencing reads were assessed for quality using FastQC, then mapped to mouse strain C57BL6/J genome assembly GRC38.p4 using the HISAT2 algorithm running on the Galaxy platform with default settings. ENSEMBLE annotation release 84 was used to identify genes, transcripts, protein-coding transcripts, exons, and rRNA for feature counting. Feature counts corrected for genomic DNA contamination were obtained using SeqMonk,; read counts for genes were determined as counts mapped to the union of all exons for all transcript isoforms of a given gene. [...] Differential expression analysis and generation of the corresponding data plots were performed using the DESeq. 2 package on the Bioconductor platform using default settings, excepting that the alpha (FDR) threshold was set to 0.01 and a fold change threshold minimum of 2 was imposed. The Bonferonni correction for multiple comparisons, defined by p < 0.05 /m, where m is the number of genes sequenced by RNAseq, was set at 2.6898 × 10−6 for judging statistically significant differences in gene expression.Gene ontology (GO) enrichment analysis was carried out using Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8 beta (2016 knowledgebase) to identify functional classes of genes by biological processes. Biological processes GO terms were queried and significant terms were identified as those with p-values ≤ 0.05. The top ten biological processes for each time point were chosen after removing those processes with greater than 75% redundancy of listed genes when compared to a more statistically significant biological process. The R package GOplot 1.0.2 was used to generate data plots with GO enrichment using default settings. Differentially expressed genes identified within the top ten most significant biological processes were subsequently filtered by p-value and base mean greater than 200, choosing the top 50 genes with lowest p-value and average read count greater than 200. Functional protein association networks of these differentially expressed genes were developed using STRING v10.0 and strength of protein association was determined by confidence score. […]

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