Barentsia gracilis transcriptome
Specimens were preserved in RNAlater overnight at room temperature or 4°C followed by storage at ‑80°C or fresh tissue was frozen at -80°C. Total RNA was extracted using the RNAqueous Micro kit (Ambion) without a DNase digestion, the RNeasy Micro kit (Qiagen) with on-column DNase digestion, or TRIzol (Invitrogen) followed by purification using the RNeasy kit (Qiagen) with on-column DNase digestion. RNA concentration was estimated using a NanoDrop 2000 (Thermo Scientific) and RNA quality was evaluated on a 1% SB agarose gel. For most libraries, first-strand cDNA was synthesized from 1 µg of total RNA. However, for some very small samples, the eluted RNA was too dilute to measure using the NanoDrop or visualize with agaorse gel electrophoresis. In cases where less than 1 µg of total RNA was available, 1 µl of RNase-OUT (Invitrogen) was mixed with all of the eluted RNA, this mixture was vacuum-centrifuged to a volume of 3 µl, and all 3 µl were used to make cDNA. First-strand cDNA synthesis was performed using the SMART cDNA library construction kit (Clontech) as per the manufacturer’s instructions except that the provided 3’ oligo was replaced with the Cap-Trsa-CV oligo as per Meyer et al. (2009). Full-length cDNA was then amplified using the Advantage 2 PCR system (Clontech) using the minimum number of PCR cycles possible (usually 17-19) and sent to the HudsonAlpha Institute for Biotechnology (Huntsville, AL, USA) for paired-end (PE) library preparation and Illumina sequencing. Each library was sequenced using approximately one-sixth of an Illumina HiSeq 2000 lane using the 2 X 100 bp PE chemistry.