Illumina Genome Analyzer II paired end sequencing; Sponge development
Sponge development
Adult Sycon ciliatum specimens were collected from fjords located near Bergen, Norway, and transported to laboratory in sea water collected at the place of their growth, with temperature between 14 and 18 degrees Celsius. They were gradually (1-3 hours) acclimatized to ambient temperature, avoiding temperatures exceeding 20 degrees. Field-collected water was gradually exchanged for sterile filtered natural sea water by replacing 1/4th to 1/3rd parts with the filtered water every 20-30 minutes 3-4 times. With aid of dissecting microscope, the samples were separated from the algae and cleaned from macroscopic contaminations by gently blowing water at them. Young syconoid sponges were immediately processed for RNA-isolation by placing in the lysis buffer from the AllPrep DNA/RNA Mini Kit (Qiagen). To obtain larvae, sponges containing larvae were kept in plastic cups with a pellet of 1-Hexadecanol (to avoid trapping and damage of the larvae by surface tension) until the larvae were released (1-4days). The larvae swimming near the surface were poured into clean Petri dishes. For RNA isolation, the larvae were kept in the petri dishes, including a pellet of 1-Hexadecanol, until settlement and metamorphosis, which took 6-10 days to complete, and which was periodically monitored under dissecting microscope. Once majority of specimens reached the targeted stage of metamorphosis, specimens in significantly different stage of development were removed and the remaining specimens were used for RNA isolation. Total RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen) according to manufacturers instructions. As the juvenile sponges were attached to the bottom of the petri dishes, the water was replaced with the lysis buffer and juveniles scraped of the bottom using cell culture scraper before transferring to 1.5 ml tubes and disruption. Isolated RNA was analysed on agarose gels and quantified by Nanodrop, some samples were additionally tested using Bioanalyzer. The libraries were prepared according to Illumina instructions for RNA sample preparation: TruSeq RNA V2.
Experimental Factor: developmental stage
metamorphosis stage i
LIBRARY_STRAND
not applicable