RNA Extraction and Sequencing of Daphnia

We extracted RNA using the TRI Reagent Protocol (Sigma-Aldrich, USA) from samples preserved in RNAlater (Qiagen, USA). Any remaining genomic DNA was removed using the TURBO DNA-free Kit (Invitrogen, USA). Each treatment included five biological replicates, with each replicate comprised of 35 clonal Daphnia individuals. Extracted RNA was stored in -80°C until sequencing. Quality of the isolated RNA was assessed using RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and mRNA-seq libraries were constructed using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations. A total amount of 1 μg RNA per replicate was used as input material for RNA-seq library preparations. Samples that did not reach 1 μg RNA were pooled with a biological replicate grown under the same conditions, a process described by Takele Assefa et al. [34 (link)], leaving 18 replicates from Blue Lake and 16 replicates from Gardisky Lake. Index codes were added to attribute sequences to each sample and all 34 libraries were clustered on a cBot System using the PE Cluster kit cBot-HS (Illumina). After generating the clusters, libraries were sequenced using the Illumina HiSeq 2000 platform and 150 bp paired-end reads were generated.