GSM2411602: As 0.1 mM 4; Danio rerio; OTHER
DNA was extracted from the exposed pools of embryos using the Gentra Puregene Tissue kit (Qiagen, Hilden, Germany). Genomic DNA (2.5 micrograms) was spiked with 8 methylation standards with defined methylation levels of ranging from 0 to 100%. Spiked DNA was digested with 100 units of SmaI endonuclease (NEB) for 3 hours at 25°C. SmaI cuts only unmethylated sites leaving blunt ends. The enzyme is completely blocked by CpG methylation. Subsequently, 100 units of XmaI endonuclease (NEB) were added and the digestion was continued for an additional 16 hours at 37°C. XmaI can cut both unmethylated and CpG methylated sites (CCmeCGGG) leaving 5’CCGG overhangs. The unmethylated sites thus have the GGG signature whereas the methylated sites have the CCGGG signature at the 5’ ends of the restriction fragments. Digested DNA was supplemented with dCTP, dGTP and dATP (0.4 mM final concentration of each), 15 units of Klenow Fragment (3'>5' exonuclease deficient) DNA polymerase (NEB) and incubated for 30 minutes at 37°C. This step filled in the recesses at 3' DNA ends created by XmaI digestion and added 3' dA tails to all fragments. Restriction fragments with methylation-specific signatures were ligated to NEBNext Illumina adapters and amplified using NEBNext® Multiplex Oligos for Illumina®, KAPA HiFi HotStart ReadyMix PCR Kit and 12 PCR cycles.