title: Combining profiling of transcriptome and DNA methylome to search for candidate genes as epigenetic markers in genetically selected sea bream subjected to nutritional programming
design: Liver DNA was extracted using the Quick-DNA Mini-prep Plus Kit (Zymo Research, Irvine, CA, USA) following the manufacturers instructions. The quantity and quality of DNA were assessed by a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and DNA integrity was analysed in a 1% agarose gel. For the methyl-GpG-binding domain sequencing (MBD-seq) analysis, 300 ng of DNA from each sample was fragmented to 200-550 pb using the methylation-insensitive restriction enzyme MseI (New England Biolabs, United States), which recognizes genomic TTAA sites, typically found outside of CGIs. The enzyme action and inactivation temperatures, as well as its actuation times and concentration were fixed according to manufacturers indications. Products were obtained using AMPure beads () and checked and quantified with Picogreen (Invitrogen, Carlsbad, United Stated). Fragmented DNA was then submitted to methylation enrichment using the MethylCollectorTM Ultra kit (Active Motif, Carlsbad, CA, United States). Briefly, methylated DNA was captured from 75 ng of fragmented DNA via binding to the methyl-CpG binding domain of the MBD2 protein. Illumina MBD-seq libraries were prepared from 15 ng of methylated DNA fragments using the NEBNext Ultra II DNA Library Prep Kit (Illumina Inc. San Diego, CA, USA) according to the manufacturers instructions. All libraries were sequenced on an Illumina NovaSeq 6000 sequencer as a 1  150 nucleotides single-end (SE) read format, according to the manufacturers protocol.