title: GSM9294266: GC-AS-10307-5B,Baseline,5,male; Homo sapiens; RNA-Seq
library_construction_protocol: Skeletal muscle tissue derived from the human vastus lateralis (33 ± 6 mg) was added to 2 ml homogenizing mix tubes (Fisherbrand™, Fisher Scientific) containing 600 μl Buffer RLT (Qiagen) with added 1% β-Mercaptoethanol (β-ME) before transferring to a tissue homogenizing instrument (MagNA Lyser; Roche) for subsequent homogenization (3 × 45 s at 6000 rpm, placing samples on ice for 5 mins between each disruption). Following homogenization, RNA was purified using a RNeasy® Mini kit (Qiagen) and treated with DNase (RNAse-Free DNase Set, Qiagen) as per the manufacturer's instructions. RNA quantity and purity for human samples (128 ± 175 ng/mg tissue, 1.9 ± 0.1 A260/A280 and 2.1 ± 0.2 A260/A230) samples were quantified via spectrophotometry (QIAxpert®, software version 2.4, Qiagen) and concentrations confirmed via fluorometry (Qubit®, Qiagen). RNA integrity prior to sequencing was confirmed via Agilent Bioanalsyer 2100 with RIN scores 7.22 + 0.36. 100 ng of total RNA from each sample was used to prepare mRNA libraries using the NEBNext mRNA isolation kit in conjunction with the NEBNext Ultra II Directional RNA Library preparation kit (New England Biolabs, Massachusetts, USA), and samples were randomised before preparation. Fragmentation of isolated mRNA prior to first strand cDNA synthesis was carried out using incubation conditions recommended by the manufacturer for an insert size of 300 bp (94°C for 10 minutes). 13 cycles of PCR were performed for final library amplification. Resulting libraries were quantified using the Qubit 4.0 spectrophotometer (Life Technologies, California, USA) and average fragment size assessed using the Agilent 4200 Tapestation with D1000 screentape (Agilent Technologies, Waldbronn, Germany). A final sequencing pool was created for each project using equimolar quantities of each sample with compatible indexes. 59 bp paired-end reads were generated for each library using the Illumina NextSeq 2000 in conjunction with the NextSeq 2000 P3 100 cycle kit (Illumina Inc., Cambridge, UK).