title: Illumina HiSeq 4000 paired end sequencing; Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing
design: Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing
library_construction_protocol: Mice were killed by a Schedule one procedure and the AT muscle dissected and snap frozen. RNA extraction was performed using the RNeasy Fibrous Tissues kit (Qiagen, UK) as per manufacturer's instructions. Approx. 30mg of tissue was lysed and homogenised using an ultra turrax (IKA Homogeniser, Sigma, UK) in buffer RLT before proteinase K digestion steps, total RNA bound followed by several washing steps and DNase digestion and total RNA eluted. Total RNA integrity was confirmed using the Bioanalyzer (Agilent Technologies, USA). Ribosomal RNA (rRNA) was depleted from RNA samples by using the Ribo-Zero™ rRNA Removal Kit (Epicentre, USA) in accordance with the instructions of the manufacturer. DNA free total RNA was selected for poly A using NEB Next® Poly(A) mRNA Magnetic isolation Module. RNA–Seq libraries were prepared from the enriched material using the NEBNext®Ultra™Directional RNA Library Prep Kit for Illumina®#E7420. Libraries were purified using AMPure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser. The quantity was completed using a Qubit® dsDNA HS Assay Kit, while the quality and average size was assessed using the High Sensitivity DNA Kit. Subsequently, qPCR assay, designed to specifically detect adapter sequences flanking the Illumina libraries, was performed using an Illumina® KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, USA). The RNA libraries were sequenced on an Illumina® HiSeq 4000 platform with version 1 chemistry using sequencing by synthesis (SBS) technology to generate 2 x 150bp paired-end reads. Sequencing was performed at the Centre for Genomic Research, University of Liverpool (https://www.liverpool.ac.uk/genomic-research/).