title: RNA Seq of Brienomyrus brachyistius BB-HT-08 Control Electric Organ
design: Total RNA was extracted using Qiagen RNeasy Plus Universal mini kit following manufacturers instructions (Qiagen, Hilden, Germany). Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA poly-A selected sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturers instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 min at 94C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation and quantified using Qubit 2.0 Fluorometer as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on flow cells. After clustering, the flow cells were loaded on to the Illumina HiSeq instrument (4000 or equivalent) according to manufacturers instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.