title: GSM5830437: noTraining_noNR_noPT_5; Mus musculus; RNA-Seq
library_construction_protocol: 1 ml TRIzol reagent (Life Technologies, USA) was added to each tissue and each sample was homogenised through the use of steel beads and a Tissuelyzer II (Qiagen, Germany). Beads were collected, and 200 μl chloroform was added and mixed thoroughly. After a 5 min incubation, the tubes were spun at 12,000 g for 15 min in a precooled centrifuge. The aqueous supernatant was mixed in two volumes of 99% ethanol in new tubes, and stored at -20℃ overnight. These samples were then added to RNA columns from an RNeasy kit (Qiagen, Germany) and isolated according to manufacturer's instructions. We included the optional DNase digestion steps. RNA concentration was measured by Nanodrop (Thermo Fisher Scientific, USA), and RNA integrity was tested on a Bioanalyzer instrument (Agilent Technologies, USA) according to manufacturer's instructions. All RIN values were above our preselected minimum of 8. Messenger RNA sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA protocol (Illumina, USA). Poly-A containing mRNAs were purified by poly-T attached magnetic beads, fragmented, and cDNA was synthesised using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, USA). cDNA was adenylated to prime for adapter ligation and after a clean-up using AMPure beads (Beckman coulter, USA), DNA fragments were amplified using PCR followed by a final clean-up. Libraries were quality controlled once more using a Bioanalyzer instrument (Agilent Technologies, USA) and subjected to 52-bp paired-end sequencing on a NovaSeq 6000 (Illumina, USA). A total of 1.6 billion reads was generated.