GSM5526427: J1 POST; Homo sapiens; RNA-Seq
Pre- and post-bed rest subcutaneous adipose tissue samples were obtained from ~5 cm lateral to the umbilicus with a 14G needle using the needle aspiration method (28) under local anaesthesia (1 % Lidocaine hydrochloride containing 0.005 mg/ mL adrenaline; Xylocaine; Dublin, Ireland). Adipose tissue was taken following blood draws 6 days prior to the start of bed rest and following 56 days of bed rest. Adipose samples were cleared of visible connective tissue, blood, and vasculature prior to washing the remaining tissue with 0.9 % NaCl solution (B.Braun; Sheffield, UK) over a single-use sterile 100 µm gauze to minimise blood contamination. Tissue was snap frozen in liquid nitrogen until analysis.  Total RNA (including microRNAs) was extracted using miRNeasy Mini Kit (Qiagen; Crawley, UK) according to manufacturer instructions. Following RNA isolation, samples were DNase-treated in DNase I supplemented with 10 % DNase buffer (Qiagen; Crawley, UK) for 10 minutes at room temperature, and purified as previously described (30). Thirty microlitres of RNA at a set concentration of 2.1 µg/ 30 µL were used for transcriptomic analysis. RNA-sequencing was performed on polyA-enriched 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 4 and 8.4. Input material was normalised to 200 ng prior to library preparation. 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.