Computational protocol: EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex

Similar protocols

Protocol publication

[…] Total RNA was isolated from the presumptive interscapular BAT depots of wild-type and Ehmt1myf5 KO mice at P1 or from the interscapular BAT depots of wild-type and Ehmt1adipo KO mice at 12-week-old. RNA-sequencing libraries were constructed from 50 ng of total RNA from the Ehmt1adipo KO and Ehmt1myf5 KO BAT using Ovation RNA-sequencing system v2 kit (NuGEN). mRNA was reverse transcribed to cDNAs using a combination of random hexameric and a poly-T chimeric primer. The cDNA libraries were subsequently amplified by single primer isothermal amplification (SPIA) method using Ultralow DR library kit (NuGEN) according to manufacturer’s instruction. Qualities of the libraries were determined by Bioanalyzer (Agilent Technologies). Subsequently, high-throughput sequencing was performed using a HiSeq 2500 instrument (Illumina) at the UCSF genomics core facility. RNA-sequencing reads for each library were mapped independently using TopHat version 2.0.8 against the UCSC mouse genome build mm9 indexes, downloaded from TopHat website (http://tophat.cbcb.umd.edu/igenomes.shtml). The mapped reads were converted to FPKM (fragments per kilobase of exon per million fragments mapped) by running Cuffdiff 2 on the alignments from TopHat and the UCSC coding genes to estimate gene and isoform expression levels. Based on the list of genes that showed significant difference (P <0.05, the Delta method-based hypothesis test) from the RNA-sequencing data, enrichment of the Gene Ontology (GO) biological process terms (GO FAT category) was analyzed using the Gene Set Enrichment Analysis (GSEA) program, according to the method described by the previous paper . RNA-sequencing reads have been deposited in ArrayExpress (www.ebi.ac.uk) under accession number E-MTAB-1704. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Databases iGenomes
Application RNA-seq analysis