control sequencing of Illumina mRNA Cucumaria geogiana
Collection  The animals were collected at Rothera Research Station, Adelaide Island, Antarctic Peninsula (67o 34׳ 07״ S, 68o 07׳ 30״ W) by SCUBA divers during the austral summer at depths of 10-15m. Animals were acclimated in the flow-through aquarium facility at Rothera for 2 weeks prior to the experiment and the control animals (n = 10) were maintained in the same system. Temperature in the control tanks (500 l volume) was maintained at external ambient sea temperatures using flow-through water pumped directly from the sea at rates in excess of 20 l min-1. The whole aquarium system was held in a controlled temperature room where air temperatures were set at 0.5-1.0°C.     Thermal trials  Animals (n = 10) were placed in 75 l internal volume tanks with hollow walls, through which water was pumped from a temperature-controlled unit. The whole system was placed inside a temperature-controlled room and the system held at 0ºC. After transfer of the animals to the experimental system animals, temperatures were raised by a degree an hour. Animals were checked every 30 mins and were sampled when they responded poorly to tactile stimuli. The upper lethal temperatures of these species had previously been determined and the animals were sampled at least 1°C below this temperature to ensure that they had not reached their upper lethal temperature.    Animal and tissue sampling  After the thermal treatment, the animals were quickly measured (wet weights ± 0.001g) before being snap frozen in liquid nitrogen. The whole animal was frozen, but the end portion of the animal (cross-section of body wall). A set of controls kept at 0ºC (n = 10) were sampled at the same time. Tissues were stored at -80ºC until required.    RNA extraction protocol  RNA was extracted from n=5 for each set of control and treated animals using TRI reagent (Bioline) and purified on RNeasy mini-columns (Qiagen) according to manufacturer’s instructions. RNA was quantified using an Agilent Technologies Tape Station 2200. These were the same animals that had been used in the metabolomics analyses. A more limited sampling protocol was employed for the RNA extractions, based on the preliminary metabolomics results. The RNAs were subjected to RNA-Seq on an Illumina Hi-Seq 2000 at The Genome Analysis Centre (TGAC), Norwich.