title: Local adaptation patterns in red coral populations subjected to climate change using transcriptomics
design: Local adaptation patterns in red coral populations subjected to climate change using transcriptomics
library_construction_protocol: Six adult colonies were randomly collected from two populations at different depths with contrasting thermal regimes along the Catalonian coast (Spain; Figure 1a). The first population was sampled (N=3 colonies) at 15 m depth at Cap Castell (3°13'10.233\" N, 42°4'56.276\" E), while the second population was sampled (N=3 colonies) at 48 m depth at Pota del Llop (3°13'33.787\" N, 42°2'52.736\" E). The six experimental replicates from each individual were divided into two treatments (3 replicates each): a control (18°C) and a stress (25°C), following a common garden experimental set-up for ten days. For the stress treatment, the temperature was increased stepwise from 18°C to 25°C over three days and maintained at 25°C until the end of the experiment. Fragments of C. rubrum were sampled threefold for RNA sequencing during the initial day of the experiment (T0), day 5th (T5), and day 10th (T10) in the two treatments (control and stress) and stored in RNAlater at -20°C. RNA extractions were conducted using the RNAeasy Mini Kit protocol (Qiagen) with a modification of the lysis step that involved smashing the tissue with a sterile cane in TRIzol™ (Thermo-Scientific). The resulting RNA was quantified using the Qubit® RNA BR Assay Kit (Thermo Fisher Scientific), while quality was assessed using the RNA Integrity Number (RIN), obtained on a Fragment Analyzer system, using the DNF-471 RNA kit (Agilent). Samples with a RIN > 6 were retained. mRNA-focused sequencing libraries were prepared using the KAPA Stranded mRNA-Seq Kit for Illumina Platforms (Roche), following manufacturer's recommendations, starting with 500 ng of total RNA. Illumina-compatible adapters with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated to the RNA fragments. The ligation products were enriched via 15 cycles of PCR. The final libraries were validated using the DNA 7500 Assay on an Agilent Bioanalyzer 2100 system.