Computational protocol: Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion

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Protocol publication

[…] RNA-seq was performed in RNA samples that were extracted from EndoC-βH1 cells transfected with either siNTP, siPRC1, siSRR, siZFAND6, or siZFAND3, from at least three independent transfections. RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. The libraries were sequenced using the HiSeq 4000 (Illumina). A mean of 65 million paired-end reads of 75 bp were generated for each sample. More than 90% of the reads for each library were effectively mapped to the hg19 human genome assembly using TopHat2 . Subsequently, both quantification and annotation of the reads were performed using Bioconductor package Rsubread . Finally, the differential gene expression analyses (i.e. EndoC-βH1 cells transfected with siNTP versus EndoC-βH1 cells transfected with either siPRC1, siSRR, siZFAND6, or siZFAND3) were performed using Bioconductor package DESeq2 . The differentially expressed genes were subjected to Ingenuity Pathway Analysis (Qiagen, Hilden, Germany) to decipher the major biological pathways, networks, and diseases emphasized by the significantly deregulated genes (with a p-value < 0.05). […]

Pipeline specifications

Software tools TopHat, Subread, DESeq2, IPA
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus