2.3 Sample cultivation, collection, and RNA isolation
Samples for RNA-seq were first cultivated as batch cultures in 1L glass flasks containing WC medium at 20 °C under 16:8 light:dark photoperiod. The same food combination of two algae species that used in life-table experiments was provided ad libitum every two days for two weeks before the RNA-seq experiment. For each species and temperature, we sub-sampled the initial stock into new flasks to create four replicates of 200ml each, containing more than 1000 individuals. We placed the flasks into water-baths adjusted to the experimental temperature and heat-exposed the rotifers for 4 hours. Experimental temperatures were selected according to the life-table results in order to represent control (20 oC; temperature in which both clones were acclimated), mild heat treatment (23 oC for B. fernandoi; 26 oC for B. calyciflorus s.s.; intermediate temperature between control and high heat exposure), and high heat treatment (26 oC for B. fernandoi and 32 oC for B. calyciflorus s.s.; temperature in which the population growth rate starts to decline) (Figure 1). For specimen collection we filtered each replicate (200ml) through a 30μm sieve, re-suspended what remained on the filter in WC medium, and centrifuged it at 2000 ×g for 10 minutes to pellet phytoplankton and other debris, before transferring the rotifers (remaining in the supernatant) into 1ml of TRIzol®LS and storing them at -80oC until RNA extraction. For RNA extraction, we used four replicates from each temperature and species with the exception of B. fernandoiat mild heat, where two replicates were used. Each replicate, comprised approximately of 1000 individuals, reared in independent flasks of 200ml.
Samples in TRIzol®LS were homogenized using a Tissue Lyzer (4min, 50HZ) and were incubated overnight at room temperature. A total of 500μl of chloroform was added to each sample, and samples were centrifuged for 15 minutes at 4°C to facilitate phase separation. We then transferred the colorless, upper aqueous phase into an RNeasy® Mini Kit column (Qiagen, Germany) and proceeded to RNA precipitation according to the manufacturer’s instructions. A double elution step was performed with 20μl RNase-free water each. Total RNA concentration was estimated using a NanoDrop 1000 spectrometer (ThermoFischer Scientific, Germany). Quality of total RNA was examined using Agilent Bioanalyzer 2100 (Agilent Technologies, USA).
For transcriptomic library preparation, mRNA was enriched from 3μg of total RNA with poly (A) capture using NEXTflex™ Poly (A) Beads, and strand-specific libraries were built using NEXTflex™ Rapid Illumina Directional RNA-Seq Library Prep Kit (Bioo Scientific, USA) according to manufacturer’s instructions. Final elution was performed in 16μl of elution buffer and a PCR amplification of 14 cycles was performed. Libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Germany) and quality control was performed using Agilent Bioanalyzer 2100 (Agilent Technologies, USA).