RNA-Seq of Daphnia magna: pool of 5 adult female whole individuals
To address EPA-dependent expression profiles, we fed Daphnia magna different diets in a comparable experimental design.  Two different EPA-free basal food sources were used in the experiment: the green alga ( GA ) Acutodesmus obliquus (formerly: Scenedesmus obliquus) and the Cyanobacterium ( CY ) Synechococcus elongatus. The PUFA EPA was added via liposomes to half of the experimental groups fed with GA or CY to provide a comparable design. In addition, we also monitored D. magna fed with Cryptomonas ssp. ( CRY ), serving as a natural positive control in the experimental design. In general, neonates were fed the respective food sources until reaching maturity, i.e. the production of their first clutch of parthenogenetic eggs, and were sampled subsequently. The whole experiment of testing 5 different food sources in D. magna was performed at temperatures of 20 C and 15 C to monitor effects of the key abiotic factor, as well.In total, 10 treatment groups (2 temperatures x 5 food conditions) were prepared for sequencing with 3 replicates each. Replicate sample pools consisted of 5 specimens to ensure appropriate amounts for RNA extraction and comparability. Total RNA was extracted using the NucleoSpin RNA extraction kit (Machery und Nagel, D ren/Germany) according to the manufacturer s instructions. In addition, traces of remaining genomic DNA were removed using the Turbo DNAfree Kit (Invitrogen, Karlsruhe/Germany) in line with the manufacturer s manual. The quality of the isolated RNA was monitored using capillary electrophoresis on a Bioanalyzer (Agilent Technologies, Santa Clara/USA). Only samples with OD280/OD260   2.0, OD280/OD230   2.0 and RIN   8.0 were used for sequencing. Clean-up of mRNA, cDNA library construction as well as sequencing was conducted at the University of Cologne s  Cologne Centre for Genomics . Sequencing was done using the Illumina HiSeq4000 platform in the paired end 75 bp-mode and one lane for the above described 15 sample pools (5 treatment groups with 3 replicates). Raw sequencing data were clipped and trimmed using Trimmomatic to remove bias of sequencing adapters and low quality regions at the beginning and end of sequences.