title: Patella vulgata transcriptome after heat stress; control group
design: Twenty-two individuals of P. vulgata with maximum lengths between 30 and 35 cm were collected, and moved into a recirculating aquarium within one hour of sampling, and acclimated for a full week under non-stressful conditions before the actual experiment. Temperatures in the acclimation period varied between 16 C in water (during high1 tide) and 18 C in air (during low tide). Tidal cycle was maintained at 6 h emersion and 6 h immersion, and photoperiod at 12 h light and 12 h dark. Temperatures selected for the experiments mirrored natural conditions.  Environmental conditions in the lab (water and air temperatures, infra-red radiation, visible light and tides) were regulated by a custom-made sensor/actuator controller based on Arduino (Arduino UNO R3, www.arduino.cc).Two control groups were used: five individuals sampled at the end of the acclimation period (control group for the one-day treatment), and six individuals maintained at the acclimation conditions for three more days before being sampled (control group for the three-day treatment). The remaining individuals were submitted to one-day or three-days of thermal stress during emersion (i.e. low tide). The one-day treatment consisted of exposing six limpets to a single heating event peaking at 30 C (with 4 C/h during heating and -5.6 C/h during cooling). In the three-day treatment, five limpets were exposed to an increasing thermal stress each day. Peak temperatures reached 37 C on day 1, 41C on day 2 and 43C on day 3 (with realistic heating rates of 5.25 C/h, 6.25 C/h and 5.78 C/h and cooling rates of -8.4 C/h, -10 C/h and -12.44 C/h, respectively). After the heat stress treatments, the foot of each individual was dissected and flash frozen in liquid nitrogen, and held at -80 C until RNA extraction.Total RNA (RNA) was extracted by homogenizing foot tissue (~50mg average wet mass) in 1mL Trizol while shaking with two 3 mm stainless steel beads at maximum speed in a TissueLyser II (QIAGEN 85300) for 20min at room temperature, followed by a phenol:chloroform-based RNA extraction. Total RNA concentration and purity were determined spectrophotometrically using a Qubit fluorometer (Invitrogen, CA, USA) and RNA integrity was confirmed with a BioAnalyzer 2100 RNA chip (Agilent Technologies, CA, USA). Individual cDNA libraries were constructed for each RNA extract using an Illumina TruSeq stranded mRNA LT kit (RS-122-2101) with adapter sets A and B, following the manufacturers protocol.All n=22 cDNA libraries were paired-end (PE) sequenced (150 bp reads) in the Vincent J. Coates Functional Genomics Laboratory at UC Berkeley, USA on a single lane of an Illumina HiSeq4000 platform. Libraries were qPCR quantified to ensure that equal concentrations of each sample were added to the multiplexed lane.