2.2 Tissue sampling
For each condition, the four left gill arches were sampled in 5
fish/salinity for epigenetic analysis and stored at -80°C. In the same
fish, the four right gill arches were sampled for transcriptomic
analysis. They were immersed into RNAlater (Qiagen, Mississauga, ON,
Canada) overnight at 4 °C and stored at −80 °C for further analyses. The
experiments were conducted according to the guidelines of the European
Union (directive 86/609) and of the French law (decree 87/848)
regulating animal experimentation. The experimental design has been
approved by the French legal requirement concerning welfare of
experimental animals (APAFIS permit no. 9045-201701068219555).
2.3 Genomic DNA
extraction
Genomic DNA (gDNA) was isolated from a pool of the four sampled gill
arches of each juvenile using a Maxwell Blood®16 DNA purification kit
(Promega) with the following modifications: before adding proteinase K,
gill arches were homogenized with 300 μl of lysis buffer and crushed for
30 seconds at room temperature with a laboratory mill and stainless
steel beads (Mixer Mill MM 400, Retsch, Germany) and then briefly
centrifuged for 10 seconds. Then the manufacturer’s instructions were
followed for further gDNA extraction. Purity and concentration of gDNA
was estimated using the Qubit ™ dsDNA BR Assay Kit and the Qubit ™
Fluorimeter (Thermo Scientific, Waltham, MA, USA).
2.4 Whole-genome bisulfite
sequencing
(WGBS)
Bisulfite-conversion, library construction and sequencing (Paired-end
reads - 150 bp) were performed by Génome Québec (Montréal, Canada) on an
Illumina HiSeq X (Illumina, San Diego, CA, USA). Quality of the metrics
are indicated in Table 1S. Reads quality analysis and alignments were
performed on the local Galaxy platform (http://bioinfo.univ-perp.fr).
The quality of the reads was checked using FastQC (version 0.11.8,
Andrews 2010), and MultiQC (version 1.9, Ewels et al., 2016) was used to
aggregate fastQC results into a single report. Phred scores were higher
than 25 for more than 90% of the reads’ length for all the sequences.
FastQ reads were trimmed by Trim Galore (version 0.6.3, Krueger 2012)
based on Phred score below 20 and the adaptors sequences were removed
from reads. Trimmed reads were mapped with Bismark (version 0.22.1,
Krueger et Andrews 2011) and aligned to the reference genome of European
sea bass (Tine et al. 2014) providing global methylation percentages per
genomic context (CpG, ChG and CHH). Methylation was called using Bismark
Meth. Extractor tool which generated BED files.