title: RNAseq of Notothenia coriiceps: normoxic brain
design: Total RNA was extracted from tissues using Qiagen RNeasy Plus Universal mini kit following manufacturers instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 Fluorometer and RNA integrity was evaluated using an Agilent TapeStation 4200. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and following manufacturers instructions. Briefly, mRNAs were initially enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 C. First strand and second strand cDNA were subsequently synthesized. The cDNA fragments were end repaired, adenylated at 3ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated with an Agilent TapeStation and quantified by using Qubit 2.0 Fluorometer, as well as by quantitative PCR. The sequencing libraries were clustered on a flow cell. After clustering, the flow cell was loaded on the Illumina instrument according to manufacturers instructions. The samples were sequenced using a 2x150bp Paired End  configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data generated by the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.