Mirnaeasy mini

Frozen liver sections were homogenised (Polytron, PT-2100, Kinematica, CH) in TRIzol (ThermoFisher Scientific, USA). The aqueous phase was separated via chloroform extraction (Sigma-Aldritch, UK) and centrifugation (10,000 rpm, 4°C), total RNA extracted, and DNA digested (miRNAeasy mini, Qiagen, US). Libraries were constructed from 1 μg total RNA (TrueSeq Stranded Total RNA kit, Illumina, USA) and filtered for a RIN score > 8 and < 10 (Agilent Tape Station, Bioanalyzer, UK) for sequencing (HiSeq 2500, Illumina UK). All samples were handled according to manufacturer protocols. Adapters removed, sequences trimmed (bbduk.sh) and FASTQ files aligned to the rat genome (UCSC rn6 assembly).

Total RNA (100–1000 ng) was isolated from testes using miRNAeasy Mini (Qiagen) and RNA-seq libraries were constructed using TruSeq Stranded Total RNA Kit (Illumina) with Ribo-Zero following the manufacturers’ instruction. The fragment size of RNA-seq libraries was verified using a 2100 Bioanalyzer (Agilent) and concentrations were determined using Qubit (LifeTech). The libraries were loaded onto the Illumina HiSeq 3000 for 2x50 bp paired end read sequencing at the NHLBI DNA Sequencing and Genomics Core Facility. The fastq files were generated using the bcl2fastq software for further analysis.