GSM8207435
Total RNA was isolated using the RNeasy Fibrous Tissue Mini Kit (Qiagen) from gastrocnemius samples of mice fed a HFD for 6 weeks. RNA quality was verified using an Agilent 2100 Bioanalyzer, and individual samples with an RNA Integrity Number (RIN) > 8 were included in the study. Total RNA (100 ng) was used to generate barcoded RNA-seq libraries using the NEBNext Ultra II Directional RNA Library preparation kit (New England Biolabs) according to manufacturer's instructions. First, poly A+ RNA was purified using poly-T oligo- attached magnetic beads followed by fragmentation and first and second cDNA strand synthesis. Next, cDNA ends were repaired and adenylated. The NEBNext adaptor was then ligated followed by second strand removal, uracile excision from the adaptor and PCR amplification. The size of the libraries was checked using the Agilent 2100 Bioanalyzer and the concentration was determined using the Qubit® fluorometer (ThermoFisher) Libraries were sequenced on a Illumina MiSeq system to generate 51 base single reads.