title: RNA-seq of Temora stylifera: adult female spleen
design: Adult females (N = 10) of Temora stylifera were sorted from zooplankton sample collected from 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 three replicate 1.5 ml Eppendorf tubes per date, immediately frozen in liquid nitrogen after removing excess water and finally stored at -80 C for later total RNA extraction. Total RNA was extracted using the RNeasy Micro Kit, following manufacturers specification and performing on-column DNaseI treatment. The RNA was finally eluted in 15 l of RNase-free water. RNA concentration (ng/l) and purity were assessed through Nanodrop ND-1000 UV-Vis spectrophotometer. Overall RNA integrity and DNA contamination were checked by electrophoresis of at least 200 ng of RNA on a 1% agarose gel in 0.5x Tris Borate EDTA buffer (TBE) and by analysing 150-200 ng of RNA in a 6000 Nano LabChip of an Agilent Bioanalyzer 2100, which defines RNA quality as RNA Integrity Number (RIN). RIN values >8 were considered suitable for NGS analysis.  At least 2 g of extracted RNA per sample (200 ng/L) were used for library preparation, sequencing and de novo transcriptome assembly. cDNA Libraries were prepared using TruSeq RNA Sample Prep Kit (Illumina) according to manufacturers recommendations and pooled such that each index-tagged sample was present in equal-molar amounts.