GSM9431905: Gastrocnemius, KO 2; Mus musculus; RNA-Seq
Gastrocnemius muscle from 21-month old muscle-specific Nox4 knockout mice (MckCre:Nox4 fl/fl) and age-matched Nox4 fl/fl controls. Total RNA was isolated from homogenised mouse gastrocnemius muscle tissue with RNeasy mini kits (Qiagen, Hilden, Germany) and processed for library preparation (PolyA, unstranded) and sequencing on an Illumina Novaseq PE150 by Azenta Life Sciences (Burlington, MA). 1 μg total RNA was used for following library preparation. The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high  temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and  add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of  Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Illumina Novaseq instrument for sequencing using a 2x150 paired-end (PE) configuration according to manufacturer's instructions.