title: GSM2188846: FF2; Gasterosteus aculeatus; Bisulfite-Seq
library_construction_protocol: DNA was isolated from gills (an organ, directly exposed to water environment) using standard method of DNA extraction from animal tissue (phenol-chloroform). Two micrograms of genomic DNA from three-spined stickleback samples of G. aculeatus were digested using 60 U MspI (Fermentas, USA) in 50 μl at 37°С for 18–24 h, followed by QIAquick purification (Qiagen, Germany). The end of the digested DNA was repaired, and an adenine was added to the 3’ end of the DNA fragments according to the Illumina standard end repair and add_A protocol (Illumina, USA). Pre-annealed forked Illumina adaptors containing 5’- methylcytosine instead of cytosine was ligated to both ends of DNA fragments using standard Illumina adaptor ligation protocol (Illumina, USA). Ligated fragments were then separated by 2% agarose gel (Sigma-Aldrich, USA). Fragments between 170 bp and 350 bp, (includes adaptor length), were selected and cut from the gel. DNA from gel slices were purified using Qiagen Gel Extraction Kit (Qiagen, Germany). The sodium bisulfite treatment and subsequent clean-up of size selected DNA was performed with the EZ DNA MethylationTM Kit (ZymoResearch, USA) according to the manufacturer’s instructions. The bisulfite-treated DNA fragments were amplified using PCR and the following reaction: 5 μl of eluted DNA, 1 μl of NEB PE PCR two primers (1.0 and 2.0) and 45 μl Platinum PCR Supermix (Invitrogen, USA). The amplification conditions were as follows: 5 min at 95°C, 30 s at 98°C then 15 (10 s at 98°C, 30 s at 65°C, 30 s at 72°C), followed by 5 min at 72°C. The PCR reaction was purified by MinElute PCR Purification Kit (Qiagen), and final reduced representation bisulfite library was eluted in 15 μl EB buffer. The concentration of the final library was measured using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA).