GSM5335441: Old_E5_SkeletalMuscle; Homo sapiens; RNA-Seq
Skeletal muscle was obtained from the Vastus Lateralis of the dominant leg under local anaesthesia using the Bergström technique (Bergström et al., 1967). Tissue partitioned for transcriptomic analysis was snap-frozen in liquid nitrogen and stored at −80 oC until analysis.     Adipose tissue Subcutaneous adipose tissue samples were obtained from ~5 cm lateral to the umbilicus with a 14G needle using the needle aspiration method (Travers et al., 2015) under local anaesthesia (1 % Lidocaine hydrochloride; Hameln Pharmaceuticals; Gloucester, UK). Visible connective tissue, blood, and vasculature were removed with scissors prior to washing the remaining tissue with 0.9 % NaCl solution (B.Braun; Sheffield, UK) over a single-use sterile 100 µm gauze to further clean the sample of contaminating blood. Tissue was immediately snap-frozen in liquid nitrgen and stored at −80 oC until analysis.    Total RNA (including microRNAs) was extracted from frozen adipose tissue (~100 mg) or skeletal muscle samples (30 to 50 mg) using miRNeasy Mini Kit (Qiagen; Crawley, UK) according to manufacturer instructions. Following RNA isolation, samples were DNase treated with TURBO DNase (Thermofisher™; Leicestershire, UK) according to manufacturer instructions, followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA pellets were re-suspended in 35 µL of nucleotide-free water each (Thermofisher™; Leicestershire, UK) with 2 μL used for quality control. Thirty microlitres of RNA at a set concentration of 2.1 µg/ 30 µL was sent for RNA-sequencing. RNA-sequencing was performed on RiboZero-treated total RNA, on a HiSeq4000 (Illumina, Inc.; California, US) by the Oxford Genomics Centre (Wellcome Trust; Oxford, UK). In brief, total RNA was quantified using RiboGreen (Invitrogen; California, US) on the FLUOstar OPTIMA plate reader (BMG Labtech GmbH; Aylesbury, UK) and the size profile and integrity analysed on the 2200 or 4200 TapeStation (Agilent, RNA ScreenTape [Agilent Technologies; California, US]). RIN estimates for all samples were between 1.8 and 8.5. Input material was normalised to 200 ng prior to library preparation. Total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal Kit (Epicentre/Illumina, Human [Illumina®; California, US]) following manufacturer's instructions. Library preparation was completed using NEBNext Ultra II mRNA kit (New England Biolabs Inc.; Massachusetts, US) following manufacturer's instructions. Libraries were amplified (11 cycles) on a Tetrad (Bio-Rad Laboratories; California, US) using in-house unique dual indexing primers (Lamble et al., 2013). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to loading on the sequencer. Paired end sequencing was performed using a HiSeq4000 75 bp platform (Illumina, HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit), generating a raw read count of >38.5 million reads per sample.