GSM4655249: Seabass_PostStress_T4_K10; Dicentrarchus labrax; Bisulfite-Seq
Blood from the caudal vessel was collected. Plasma and red blood celles were separated by centrifugation at 2,000g for 10 min. Careful separation of the plasma and RBCs was performed using 200 µl pipettes. RBC extracts were heparinized (heparin sodium; Sigma-Aldrich), transferred in microtubes and conserved at -20°C in 1 ml of RNA later. Genomic DNA was extracted using the Macherey Nagel Nucleo Spin Tissue DNA kit and quantified using a Qubit fluorometer (Qubit dsDNA BR Assay Kit, Q32853, Invitrogen). EpiGBS approach adapted from van Gurp et al. (2016, Nature Methods doi: 10.1038/nmeth.3763), with main addition of a second restriction enzyme in the protocol. The couple of enzymes retained: MspI/ SfbI. A single libray was prepared: 200 ng of DNA of each sample were digested in a 40 µl reaction, using 0.25 µl MspI (NEB, 20,000 U/ml R0106S), 0.25 µl SbfI-HiFi (NEB 20,000 U/ml R3642L) and 4 µl of 10X cutsmart buffer. The reaction was run overnight at 37°C. Unique forward and reverse adapter combinations allow multiplexing samples in the library. We added forward and reverse adapters in unique combinations (1 µl of adapter, 2.5 µM), 0.5 µl T4 Ligase (NEB, 400,000 U/ml m0202L), 6 µl of 10X T4 ligase buffer and 11.5µl water were added directly to the digested DNA. Adapters were ligated for 3 h at 23°C followed by 10 min of enzyme inactivation at 65°C. After ligation, all samples were pooled and one third of the total volume was used in the following step. The mixture volume was reduced using a Qiaquick PCR purification kit (28104 Qiagen). The resulting product was cleaned a second time to ensure the removal of small fragments and adapter remnants using CleanPCR paramagnetic beads (Proteigene, CPCR-0050) with a ratio of 0.8X [sample : beads]. a nick translation was performed to repair the nick of the DNA at the restriction site and to fully methylate the hemi-methylated adapters. To do so, 19.25 µl of the concentrated and purified ligation pool were used in a 25 µl reaction, including 0.75 µl DNA Polymerase I (E. coli, 10,000 U/ml M0209S), 2.5 µl of 10 mM 5-Methylcytosine dNTP mix (Zymo Research, D1030), and incubated at 15 °C for one hour. The library was then treated with sodium bisulfite using the EZ DNA Methylation Gold Kit (D5005, Zymo Research) following manufacturer instructions to convert unmethylated cytosines to uracil, paying attention to the optimal DNA amount per reaction. After bisulfite conversion, 14 cycles of PCR (95°C for 1 min, 91°C for 10s, 65°C for 15s, 72°C for 10s and a final elongation step at 72°C for 5 min) were performed, followed by a paramagnetic bead clean up with a 0.8X ratio. The library quality was verified on an Agilent 5300 Fragment Analyzer (Santa Clara, USA). It was sequenced (paired-end, 150 bp) on one lane of a SP flow cell on an ILLUMINA NovaSeq 6000.