GSM8816786: [32] Aged (20-21 months old); Lean (12% Kcal fat diet); Mus musculus; RNA-Seq
Given the gastrocnemius is a muscle with a mixed fiber type and atrophied the most in the aged-obese (SO) animal group (Table 1), RNA from this muscle was chosen for sequencing. mRNA was isolated from the gastrocnemius muscle, where the muscle tissue was homogenized with Trizol (Life Technologies, Grand Island, NY, USA), and phenol–chloroform extraction was performed following homogenization. RNA was ethanol-precipitated and diluted in 70% of diethyl pyro carbonate-treated ethanol. Total RNA was isolated using an Invitro-gen PureLinkTM RNA Mini Kit (Invitrogen, Waltham, MA, USA; 12183018A). Total RNA was eluted in RNase-free water. RNA concentration and purity were measured using a BioTek Take3 micro-volume microplate with a BioTek Synergy HTX Multi-Mode Microplate Reader, IVD (Fisher Scientific, Waltham, MA, USA; BTS1LASI). A 260/280 nm ratio of >2 was utilized as an initial quality assessment(28). RNA concentration was assessed using a Qubit Fluorimeter, Fragment Analyzer, Nano Drop, and Microplate reader. RNA integrity and quality were assessed by quantifying the RNA integrity number (RIN) and the RNA quality number (RQN) using Agilent 2100 Bioanalyzer and Fragment Analyzer. RIN scores below 7 were excluded from subsequent analysis. Single-end mRNA sequencing was completed at the BGI Hong Kong Tech Solution NGS Lab (Hong Kong, China) using MGISeq 2000 Transcriptome for library preparation.