454 sequencing of Bugula neritina metamorphosis transcriptome fragment library
Total RNA from different developmental stages (swimming larvae, 4hours post release, 28hours post release, 52hours post release) were extracted with SV total RNA isolation system (Promega, WI, USA). The integrity of the total RNA and purified mRNA were checked with both denatured agarose gel and Nano-drop 100. Messenger RNA was further extracted with PolyA purist Mag Kit (Ambion, Austin, TX, USA). Double strand cDNA was synthesized by using a Superscript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA) with random hexamer primer. The synthesized cDNA was further purified with Phenol:Chlorform and then precipitated with ethanol. The cDNA quantity and quantity were checked with agarose gel electrophosis and Nano-drop100.