Illumina HiSeq 4000 paired end sequencing; Transcriptomic profiles of the Mediterranean sponge species Aplysina aerophoba upon wounding by the specialist grazer Tylodina perversa
Transcriptomic profiles of the Mediterranean sponge species Aplysina aerophoba upon wounding by the specialist grazer Tylodina perversa
144h-control rep 1_p
The sponge Aplysina aerophoba and the sea slug Tylodina perversa were collected by scuba diving in June 2016 and May 2017 at the Mediterranean coast of Spain (42.29408 N, 3.28944 E in 2016 and 42.1145863 N, 3.168486 E in 2017), at a depth between 2 to 10 m. After collection, animals were transported to the Experimental Aquaria Zone (ZAE) at the Institute of Marine Science (ICM-CSIC) in Barcelona (Spain). There, each sponge individual was divided into 2-3 specimens with its own osculum. Each specimen was placed into individual aquaria (6 L). All the aquaria were maintained in a flow-through system with direct intake of seawater.After 1 week acclimation, the specimens from each sponge individual were randomly assigned to one of the following three treatments (i) control: no treatment, (ii) grazing: one sea slug, which starved for 24 h, was placed in direct contact to the sponge specimen and allowed to feed freely for 24 h, and (iii) mechanical damage: the sponge specimen was clipped with a scalpel for 3 min every half hour at the first 3 hours and the last 3 hours of a 24h-period. All treatments were stopped after 24 h. This design was applied in independent experiments that differ in the sampling points after stop of treatments, to avoid repetitive sampling of the same specimen. One experiment was performed in 2016, in which samples were collected 144h after stop of the treatment . In 2017, two experiments were performed: 3h and 24h after stop of the treatment. Samples from grazing and mechanical damage treatments were collected at the scar, washed with artificial seawater, and fixed in RNAlaterTM (Invitrogen, Thermo Fisher Scientific, Germany) at 4 °C overnight. Samples were then stored at -80 °C until RNA extraction.  To extract total RNA, ca. 30 mg (wet weight) of sample was immediately homogenized in 1% ß-mercaptoethanol in RLTplus buffer (Qiagen, Germany) by using the PowerLyzerTM24 (Mobio, Qiagen, Germany). The total RNA was obtained using the AllPrep DNA/RNA Mini Kit (Qiagen, Germany) following manufacturer's protocol and eluting the RNA in 100 µL of Buffer EB (Qiagen, German). To remove DNA contamination, the extracted total RNA was treated with DNA-free DNase Treatment and Removal Reagent (Ambion, Germany). The absence of DNA was further checked by PCR amplification of eukaryotic 18S rRNA gene (Sigma-Aldrich, Germany: forward primer - ACCTGGTTGATCCTGCCAG, reverse primer - AATTATAATACGACTCACTATAGATTCYGCAGGTTCACCTAC; Stewart et al. 2010). Total RNA was checked for purity, quantity and quality by using NanoDrop 2000c Spectrophotometer (peolab, Germany), Qubit 2.0 (Life Technologies, Carlsband, CA), and ExperionTM Electrophoresis Station (Bio-Rad, Hercules, CA). Only the total RNA which was DNA-free and presented OD260/280 >1.8 (in Nanodrop) as well as RIN > 8 (Experion) was subsequently used for RNA-Seq.  Equal amounts of the total RNA (1950 ηg in 30 μl) were used for library construction with the TruSeq stranded mRNA library prep kit (Illumina, Inc., USA), including a poly-A enrichment step. Library preparation was performed at the IKMB Kiel.