Computational protocol: Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis

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Protocol publication

[…] Total RNA was prepared from EDL muscle of 8-wk-old male and MKO mice using TRIzol (Invitrogen) reagent, and poly(A) RNA was extracted with Dynabeads Oligo(dT)25 (Ambion). Sequencing libraries were prepared using the Epicentre ScriptSeq v2 and then subjected to 100-nt paired-end sequencing on an Illumina HiSeq2000 (Illumina). The libraries were quantitated by qPCR according to the qPCR Quantification Protocol Guide, and quality control was performed on an Agilent Technologies 2100 Bioanalyzer. Reads were processed from the sequencer and aligned to the Mus musculus reference genome (mm10) using TopHat v2.0.13 (). The reference genome and annotation data were downloaded from the UCSC Genome Browser (http://genome.uscs.edu). Gene annotation information was used to run TopHat with the “-G” option. After alignment of reads to the genome, Cufflinks v2.2.1 () was used to assemble aligned reads into transcripts and estimate their abundance. To correct sequence expression count bias, the “–max-bundle-frags 50000000” option was used. The transcript counts in isoform level were calculated, and relative transcript abundances were measured in FPKM (fragments per kilobase of exon per million fragments mapped) using Cufflinks, as well as gene level expression values. Raw data were calculated as FPKM of each gene in each sample using the Cufflinks software. Genes with more than two zero FPKM values were excluded. To facilitate log2 transformation, 1 was added to each FPKM value of filtered genes. Filtered data were log2-transformed and subjected to quantile normalization. The resultant values were used for DEG analysis. A fold change of two or more was deemed significant. [...] An unbiased bioinformatic analysis (GSEA; http://www.broadinstitute.org/gsea) was performed using skeletal muscle transcriptome data from BXD genetic reference population mice as described in previous studies (; ). In brief, Gdf15 expression was analyzed in skeletal muscle transcriptomes of both normal chow- and HFD-fed BXD mice (GeneNetwork accession numbers GN379 and GN380). Next, the transcriptomes of 10 HFD-fed BXD mouse lines exhibiting the largest alterations (highlighted in red and blue, ) in Gdf15 expression were subjected to GSEA. Heat maps (, left) were built using GENE-E (The Broad Institute; http://www.broadinstitute.org/cancer/software/GENE-E/). Correlation and principal-component analyses () were performed using the appropriate functions and packages in R (http://www.r-project.org). The depth of shading at the correlation matrices (corrgram) indicates the magnitude of the correlation (Pearson’s R). Positive and negative correlations are represented in blue and red, respectively. All raw data used in GSEA are available on the GeneNetwork website (http://www.genenetwork.org). Parameters for all assays were as follows. Activity: locomotor activity for 24 h (counts per hour), standard housing cage at 23 wk of age, HFD, males (n = 52, GeneNetwork record ID 17714). RER imp: RER improvement (ratio) after 10 d of voluntary exercise, treadmill at 10° incline, chow diet, males (n = 49, GeneNetwork record ID 17749). Glucose: serum glucose level (millimoles per liter) at 70 d in males fed an HFD from 4 wk (n = 32, GeneNetwork record ID 15045). Food in: food intake [g] in 24 h at 16 wk of age, metabolic cage, chow diet, males (n = 36, 24 h at 16 wk of age, metabolic cage 17633). Hep Lip: hepatic lipid (ratio of total lipid to total protein) measured by Fourier transform infrared spectroscopy in liver at 140 d in males fed an HFD from 4 wk (n = 31, GeneNetwork record ID 15095). Liver we: liver weight (grams) at 29 wk of age, chow diet, males (n = 50, GeneNetwork record ID 17767). pWAT: peritoneal adipose tissue weight (grams) at 20 wk in females and males fed an HFD from 4 wk (n = 32, GeneNetwork record ID 14981). sWAT: residual subcutaneous fat weight (grams) at 20 wk in females and males on HFD feeding from 4 wk (n = 32, GeneNetwork record ID 14992). Fat mass: total white adipose weight (grams) at 20 wk in the whole body in females and males on HFD feeding from 4 wk (n = 32, GeneNetwork record ID 14987). Bd We: body weight gain (grams) between 9 and 10 wk in females and males fed an HFD from 4 wk (n = 32, GeneNetwork record ID 14952). […]

Pipeline specifications

Software tools GSEA, GENE-E
Application CAGE analysis
Organisms Mus musculus
Diseases Deficiency Diseases