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Pipeline publication

[…] b' than 89%., The mRNA obtained from blood was sequenced using a Genome Analyzer IIx from Illumina. Total RNA was extracted from peripheral blood of each individual. The mRNA was isolated from the total RNA and, once transformed into cDNA, was fragmented by sonication. Fragments of 300bp on average were selected to construct the libraries for sequencing. The libraries to sequence were constructed using the reagents according to the indications of the manufacturer (Illumina). Single-end sequences of 35 nucleotides were produced using a Genome Analyzer IIx (Illumina). Alignment of the reads was performed in an Simple Linux Utility for Resource Management High Performance Computer server running Tophat 2.0.6 with default options (). Tophat aligns RNA-Seq reads to genomes using the Bowtie 2.0.2 alignment program (), and then analyzes the mapping results to identify splice junctions between exons., Bedtools 2.17.0 (multicov option; ) was used to count the amount of reads mapped to each gene. The Reference Sequence gene coordinates were defined using the RefFlat file from the UCSC Genome Bioinformatics Site (retrieved February 28, 2014). DESeq 1.10.1 package (), setting up fit-only as fitting method, was used to test for differential expression using gene-count data., To validate the expression data obtained by RNA-Seq, we performed quantitative reverse transcription PCR amplification of 10 of the genes that were found to have differential expression between cases and controls () on 30 randomly selected samples (15 cases before medication and 15 controls). We used pre-designed Taqman probes (Life technologies) using the manufacturer\xe2\x80\x99s instructions for LAMP3 (Hs00180880_m1), RFX2 (Hs01100925_m1), CTAG2 (Hs00535628_m1), GPR128 (Hs00262184_m1), IFI27 (Hs01086373_g1), ADAMTS2 (Hs01029111_m1), BRS3 (Hs00179951_m1), MSLN (Hs002458' […]

Pipeline specifications

Software tools TopHat, BEDTools, DESeq
Organisms Homo sapiens
Diseases Diabetes Mellitus, Glucose Metabolism Disorders, Body Weight, Overnutrition