GSM6422028: Hemispheric cohort, muscle, RML, 8 wpi, rep 2; Mus musculus; RNA-Seq
RNA was extracted from snap frozen areas using the RNeasy Plus Universal Kit (QIAGEN), following manufacturer's instructions. Quantity and quality of RNA were analyzed with Qubit 1.0 Fluorometer (Life Technologies) and Bioanalyzer 2100 (Agilent Technologies), respectively. The TruSeq RNA Sample Prep kit v2 (Illumina) was employed for library preparation. In brief, 1 μg of total RNA per sample was poly A enriched, reverse transcribed into double-stranded cDNA and then ligated with TruSeq adapters. PCR was performed to selectively enrich for fragments containing TruSeq adapters at both termini. Quantity and quality of enriched libraries were analyzed using Qubit (1.0) Fluorometer and Caliper GX LabChip GX (Caliper Life Sciences). The resulting product is a smear with a mean fragment size of approximately 260 bp. Libraries were then normalized to 10 nM in Tris-Cl 10 mM, pH 8.5, with 0.1% (vol/vol) Tween 20. Cluster generation was performed with the TruSeq PE Cluster kit v4-cBot-HS (Illumina), using 2 pM of pooled normalized libraries on the cBOT. Sequencing was performed on Illumina HiSeq 4000 paired-end at 2 × 126 bp using the TruSeq SBS kit v4-HS (Illumina).