Renilla Transcriptome Profiling and Assembly

Renilla total RNA was prepared from 2 live animals by freezing in liquid nitrogen, homogenization by mortar and pestle, and Trizol extraction. Poly-A⁺ RNA was isolated from 600 μg total RNA using the poly-A Spin mRNA isolation kit from New England Biolabs (NEB) using two consecutive oligo-dT selections following the kit protocols. 50 ng of poly-A⁺ RNA was used to construct a directional cDNA library using the NEBNext Ultra RNA library kit for Illumina following the kit instructions. The fragmentation step was performed for 13 min at 94°C. Final amplification was performed for 15 cycles and the library was purified from primers and adaptors using two consecutive Ampure bead steps before quality assessment in an Agilent Bioanalyzer which indicated a library with a peak corresponding to 262 bp.
Sequencing of the library was performed in the Illumina MiSeq. 12 million reads were obtained.
The reads were used as input for the Trinity assembly software [12 (link)] for de novo transcript assembly, resulting in 48,880 assembled RNA transcripts. The corresponding DNA sequences were generated. The entire assembled cDNA library generated from Trinity was translated in all 3 frames.