RNA-Seq of calanoid copepod: Calanus helgolandicus female
Adult females of Calanus helgolandicus were sorted from zooplankton sample collected on three different occasions during May 2012 in the Gulf of Naples, Mediterranean Sea, through oblique tow of 200 m Nansen net (113 cm mouth diameter) equipped with a 200 m filtering cod-end. Copepods were transferred to 1L jars (N=20) filled with 0.22 m filtered sea water (FSW) and enriched with either Skeletonema marinoi (45,000 cells mL-1, 1 mg C L-1), or Prorocentrum minimum (5,000 cells mL-1, 1 mg C L-1). Jars were kept at 18 C, on a 12:12 Light: Dark cycle. The algal medium was changed daily with addition of fresh FSW and either S. marinoi or P. minimum diet. After five days, the copepods were transferred to clean jars containing 0.22 m FSW for 24 h in order to allow gut evacuation, and then ten females per diet for each replicate sampling week were transferred into 1.5 mL Eppendorf tube containing 500 L of RNAlater reagent and processed according to manufacturers instructions. Total RNA was extracted using the RNeasy Micro Kit (Qiagen), following manufacturers specification and performing on-column DNaseI treatment. RNA concentration (ng/l) and purity were assessed through Nanodrop ND-1000 UV-Vis spectrophotometer (Marshall Scientific), whereas RNA integrity number (RIN) was checked on a 6000 Nano LabChip of an Agilent Bioanalyzer 2100 (Agilent Technologies). A total of 3 g of extracted RNA per sample (300 ng/L) were delivered to the Genomics Core Facility of the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany), for library preparation using TruSeq RNA Sample Prep Kit (Illumina). RNA-Seq was conducted on Illumina HiSeq 2000 platform with 50 bp paired end option.