Dataset for "The Impact of Physical Inactivity, Ageing, and Nutrition on Adipose Tissue Function"

Data collection method:

Adipose tissue samples were collected under local anaesthesia from abdominal subcutaneous adipose tissue depots, 5cm lateral to the umbilicus by needle aspiration. Skeletal muscle samples were collected from the Vastus lateralis using the Bergstrom biopsy technique . All samples were collected following an overnight fast in the rested state. Samples were briefly cleaned of visible signs of blood, weighed and immediately snap frozen in liquid nitrogen and stored at -80 degrees until analysis. Tissue samples were thawed and digested in QUIzol reagent, to extract RNA fractions. RNA was DNase treated and QC'd to assess sample quantity and quality. Samples at a set concentration were sent for next-generation sequencing at the Wellcome Trust Oxford Genomics Centre on a HiSeq4000 Illumina instrument, Deoxynucleotide triphosphate (dUTP) was incorporated into the second strand, to facilitate selective degradation of dUTP-tagged cDNA to generate a cDNA library suitable for sequencing. cDNA was end-repaired, A-tailed, and adapter-ligated. Samples also underwent uridine digestion. Prepared libraries were size-selected, multiplexed across 8 lanes, and quality controlled before paired end sequencing over 12 lanes of a flow cell. FastQ sequencing files were uploaded to the Galaxy web platform (usegalaxy.org) for quality control analysis. Raw sequencing files were splice-aligned to the GRCh38/hg38 reference genome using Hisat2 with a mapping distance <500 kb between reads. Genome annotation was performed using Ensembl against the reference genome. Expression levels (fragments per kilobase of transcript per million mapped reads (FPKM]) were estimated using Stringtie.

Technical details and requirements:

Equipment and software used: - 14G adipose biopsy needle and 10 or 50mL syringe - Bergstrom biopsy needle. - Quizol reagent. - DNase I and DNase buffer (Qiagen) - RNase/DNase free water - Phenol-Chloroform-Isoamyl alcohol 25:24:1 - Tris EDTA - Sodium acetate - nucleotide free water - Qubit - SpectroStar Nano - Agilent tapestation 4200 - usegalaxy.org - Hisat2 - Ensembl - Stringtie