Intestinal RNA-seq in mice with impairment of insulin action in GLUT4-expressing tissues

Abstract

RESEARCH AIMS

To test the hypothesis that high-fat diet fed mice with Insr deletion in GLUT4-expressing tissues have altered gene expression in the intestine compared with controls fed high-fat diet.

METHODS

RNA-seq of intestinal tissues collected from Glut4-Cre promoter-driven Insr knockout mice and controls fed high-fat diet. Deposited data are Fastq sequence files from the RNA-seq results, compressed in ‘.gz’ format. RNA-seq of intestinal tissue collected from 6 control mice and 6 Glut4-Cre promoter-driven Insr knockout mice on high-fat diet. RNA was extracted with Qiagen RNeasy Plus Mini Kit and evaluated for quantity and quality for a minimum RIN score of 7 or higher using Agilent Bioanalyzer 2100. Two hundred nanograms of total RNA per sample were used for library preparation. cDNA libraries were prepared using RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification using KAPA mRNA Hyperprep Kit (KK8581). Each library was quantified, and its quality accessed by Qubit and Agilent 2100 Bioanalyzer. Total RNA was sequenced with the 2×75 paired-end configuration on an Illumina HiSeq 4000 with an average of 30.6M reads. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).

RNA-Sequencing alignment and analysis. All sequenced libraries were then mapped to the mouse genome (UCSC mm10) using STAR RNA-seq aligner. The reads distribution across the genome was assessed using bamutils (from ngsutils). Uniquely mapped sequencing reads were assigned to mm10 refGene genes using featureCounts.