title: N. vectensis RNA-Seq Post-gastrula 2
design: Developmental and adult N. vectensis RNA sequencing was performed as follows. N. vectensis were spawned and eggs were de-jellied and fertilized as previously described. Spawning and embryo development took place at 18 C. Eggs and embryos from different stages were collected (300 per sample) in duplicate as indicated: eggs (within 30min of spawn), blastula (7.5hpf), gastrula (23.5hpf) and planula (72hpf). Eggs and embryos were collected in eppendorf tubes and centrifuged to a pellet at 21,000 x g for 1 min. All seawater was quickly removed and pellets were resuspended in 150ml lysis buffer (RLT buffer supplied by the Qiagen RNeasy kit (#74104), supplemented with -mercaptoethanol). The samples were homogenized with an electric pestle (1 min continuous drilling) and further supplemented with 200 ml of the above lysis buffer. Homogenized samples were then transferred into QIAshredder columns (Qiagen #79654) and centrifuged at 21,000 x g for 2 min. The flow throughs were supplemented with 1 ml 70 % ethanol and transferred to RNeasy columns and were processed according to the Qiagen RNeasy protocol. Quality and integrity of the RNA was evaluated using the Agilent RNA 600 pico kit (Agilent Technologies) and RNA samples were stored at -80 C until further processing. cDNA libraries were then constructed for polyA stranded sequencing. The resulting libraries were sequenced on Illumina HiSeq using paired end runs (RapidSeq- 2x150bp).