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RNA Sequencing Facilitates Quantitative Analysis of Control and GLUT1-HK2-PFKFB3 Overexpressed Transcriptomics in Mouse Skeletal Muscle

ABSTRACT: Purpose: RNA-Seq uses next-generation sequencing to analyze expression across the transcriptome, enabling to detect known or novel features and quantify RNA. The goal of this study is to compare transcriptome profiles between control and GLUT1-HK2-PFKFB3 overexpressed mouse skeletal muscle. Methods: Total RNA was isolated from the gastrocnemius muscle of chow diet-fed M;G and control mice at the age of 3-month-old using RNAiso Plus (Takara Bio). Three independent pooled samples per group were analyzed. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Fragments Per Kb of exon per Million mapped reads (FPKM) were calculated using featureCounts v1.5.0-p3. Results: We mapped about 6 million sequence reads per sample to the mouse genome and identified 54533 transcripts in the gastrocnemius muscle of control and GLUT1-HK2-PFKFB3-overexpressed mice. RNA-seq data confirmed stable expression of 10 known housekeeping genes, and 7 of these were validated with qRT–PCR. A total 664 transtripts were differentially expressed in wildtype and GLUT1-HK2-PFKFB3 overexpressed skeletal muscle with a cutoff of 2.0-fold and p < 0.05 from RNA sequencing analysis. Altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method.

ORGANISM(S): Mus musculus

PROVIDER: GSE157257 | GEO | 2022/09/01

REPOSITORIES: GEO

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Project description:Purpose: Understand the transcriptional changes and alternative splicing defects associated to the pleiotropic defects observed in mdf-1 mutants. Methods: Total RNA was extracted from 3 different biological replicates containing 12-day-old seedlings of WT and mdf-1 using the innuPREP Plant RNA kit (Analytik Jena Bio solutions). RNA quality assessment, library preparation, sequencing and bioinformatic analyses were performed by Novogene Co. Integrity and quantitation of the extracted RNA were measured using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). With 1 µg RNA per sample as input material, sequencing libraries were subsequently generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB) following the manufacturer’s recommendations. Index codes were also added to attribute sequences to each sample. Clustering of the index-coded samples was carried out on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) following manufacturer’s instructions. Afterwards, library preparations were sequenced on an Illumina NovaSeq 6000 platform and approximately 50 million 150 paired end reads per sample were generated. To ensure the high quality of the samples, only clean data was used for subsequent analyses. This was achieved after the removal of reads containing adapter and poly-N sequences and reads with low quality from the raw data. Mapping against the Arabidopsis TAIR10 reference genome was performed using the HISAT2 software. Reads Per Kilobase of exon model per Million mapped reads (RPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analyses between mdf-1 and WT were carried out using the DESeq2 R package with the Benjamini and Hochberg’s approach for adjusting P values according to the False Discovery Rate (FDR). Genes with adjusted p-value < 0.05 were considered as differentially expressed. Alternative splicing (AS) analysis was performed with the rMATS software. Events with adjusted p-value < 0.05 were considered as alternatively spliced. Results: Loss of MDF function is associated with growth defects, impaired transition to mitosis, increased endoreduplication and spontaneous cell death in meristems. This was associated with strong upregulation of stress-induced genes and down-regulation of mitotic regulators and a subset of genes required for essential developmental processes. MDF is also required for the correct splicing of numerous transcripts including transcription factors and cell cycle associated genes.

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RNA Sequencing Facilitates Quantitative Analysis of Control and GLUT1-HK2-PFKFB3 Overexpressed Transcriptomics in Mouse Skeletal Muscle

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