RNA-seq of inguinal White Adipose Tissue (iWAT) from 20-month-old WT versus KO mice
Description
To elucidate the transcriptional changes underlying the increased fat metabolism observed in aged GSNOR KO mice, we conducted a comparative analysis of gene expression profiles in iWAT from aged WT and GSNOR KO mice via RNA-seq.
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TRIzol reagent was used to extract total RNA from mouse tissue. The RNA was reverse transcribed into cDNA via the M-MLV reverse transcriptase system. The manufacturer’s instructions were followed, and oligo(dT) primers were used to specifically prime for the synthesis of cDNA from mRNA. The specific primer sequences for genes of interest are listed in Oligonucleotides. A real-time PCR system (Applied Biosystems) and SYBR Green master mix were used to perform qPCR, and 36B4 was used as the reference gene. The Ct values were analyzed, and the relative expression levels were calculated via the ΔΔCt method. RNA sequencing was conducted by Biomarker Technologies (BMK) in Beijing. RNA samples were isolated from the iWAT of the mice. The indexing and clustering of the samples were carried out on a cBot cluster generation system with the HiSeq PE Cluster Kit v4-cBot-HS (Illumina), following the manufacturer’s protocol. After cluster formation, the prepared libraries were sequenced on an Illumina platform. The RNA-seq data were analyzed via bioinformatics tools to identify differentially expressed genes between middle-aged and aged mouse iWAT samples. A functional enrichment analysis was performed to understand the biological processes and pathways associated with the gene expression changes.
Institutions
- Institute of Biophysics Chinese Academy of Sciences