Computational protocol: Regulation of Inflammatory Phenotype in Macrophages by a Diabetes-Induced Long Noncoding RNA

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Protocol publication

[…] Whole transcriptome analysis of RNA from BMMC and BMGM was performed using total RNA depleted of rRNA (RiboMinus kit, Life Technologies). Paired-end libraries were prepared, size selected, gel purified, and sequenced in our Integrative Genomics Core using an Illumina HiSeq 2000 system following manufacturer’s protocols (Illumina Inc., San Diego, CA). Reads were mapped to the mouse genome assembly mm9 using TopHat software (). Each RefSeq gene’s expression was summarized and normalized by the trimmed mean of M-value method using edgeR bioconductor R package. The total reads for each gene were then scaled by exon length to obtain average coverage. Differentially expressed genes (DEGs) were identified with coverage (≥1 in at least one sample) and fold change (≥1.5). Novel transcripts were identified using published approaches (,–), including ours (). Specifically, after de novo assembly of all the transcripts using Cufflinks (), transcripts that mapped to RefSeq genes of mouse and seven other organisms (human, chimp, rat, rabbit, orangutan, rhesus, marmoset) were first filtered out. Filtered transcripts were further assessed for potential open reading frames by using PhyloCSF and searching PfamA/B databases. Multiexonic transcripts with a score of >100 or that encoded PfamA/B protein domains were classified as novel protein-encoding transcripts. Transcripts of at least 200 bp in length containing at least two exons, having a PhyloCSF score of <100 and lacking PfamA/B domains, were defined as novel lncRNAs. Biological functions and network analysis of DEGs were performed using DAVID gene ontology and Ingenuity Pathway Analysis (IPA). TF motif analysis and enrichment were performed using TF affinity prediction (TRAP) method () and gene set enrichment analysis, respectively. […]

Pipeline specifications

Software tools TopHat, edgeR, Cufflinks, PhyloCSF, IPA
Applications RNA-seq analysis, Transcription analysis
Organisms Mus musculus, Homo sapiens
Diseases Diabetes Mellitus, Diabetes Mellitus, Type 2, Diabetes Complications
Chemicals Glucose, Palmitic Acid