RNA-seq of mus musculus brainstem
A total of 1 g of RNA per sample was used for library preparation. Samples were processed using the Illumina TruSeq Stranded mRNA Library Prep kit (Illumina, San Diego, CA) according to the manufacturers protocol. In short, mRNA was purified with Oligo-dT magnetic beads and fragmented via exposure to divalent cations and heat. First strand cDNA was then synthesised via reverse transcription using random primers. Second strand cDNA, marked with deoxyuridine triphosphate (dUTP) in place of deoxythymidine triphosphate (dTTP), was synthesised to replace the RNA template and form double stranded (ds)-cDNA. The 3 ends were then adenylated and adapters ligated to the ends of the ds-cDNA fragments. Finally, the non-uracil containing first strand cDNA fragments were selectively enriched by use of uracil-sensitive polymerase and cDNA amplification was performed. Library preparations were sequenced on the Novaseq 6000 Sequencing System (Illumina) with 100 base-pair single end reads generated. There were at least 25 million single end reads per sample. Sequencing reads were in the reverse orientation. Four biological replicates were sequenced per experimental group. Image analysis was performed in real time by the NovaSeq Control Software v1.3.0 and Real Time Analysis v3.3.3 (Illumina). The Illumina bclfastq 2.20.0.422 pipeline was used to generate the sequence data.
SUB6699098
14_SBBR_48hrs