GSM8666810: Sperm, 0day-post stripping, rep1; Cyprinus carpio; Bisulfite-Seq
Genomic DNA was extracted from 2 µL sperm samples obtained from three males using a DNA Mini Kit (Invitrogen, USA), or extracted from 50-60 mid-blastula embryos and 10-20 embryos at hatching using SDS-based methods; Total RNA was extracted from embryos using RNA isolation kits (Invitrogen, USA) in accordance with the instructions provided in the manual; Thirty embryos from each group were pooled and homogenized by a grinder with steel beads, containing 1X Cocktail, EDTA and SDS L3. The supernatant was extracted following centrifugation (25,000 g, 4°C for 15 min), and reduced with 10 mM DTT for 30 min at 37 oC. Samples were alkylated with iodoacetamide (55 mM final concentration) in the dark for 45 min. The treated samples were precipitated in cold acetone at a ratio of 1:5 for 2 h at -20 oC. After centrifugation at 25,000 g, the dry pellet was dissolved in 0.5 M triethylammoniumbicarbonate (TEAB) (Applied Biosystems). Bradford assay and SDS-PAGE were used for quality control of the protein extraction. The construction of the library and bisulfite sequencing were conducted by BGI (Shenzhen, China). Briefly, DNA was fragmented by sonication using a Bioruptor (Diagenode, Belgium) to a mean size of c. 250 bp. This was followed by the blunt-ending, dA addition to the 3'-end, and finally, adaptor ligation, essentially according to the manufacturer's instructions. Ligated DNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold kit (ZYMO). Following the administration of sodium bisulfite, unmethylated cytosine residues were converted to uracil, whereas 5-methylcytosine (5mC) remained unaffected. After the PCR amplification process, uracil residues were converted to thymine. Fragments of varying insert sizes were excised from a single lane of a 2% TAE agarose gel. The purification of the products was conducted using the QIAquick Gel Extraction Kit (Qiagen), while the amplification was achieved through PCR. Finally, sequencing was performed using the HighSeq4000 and 150-bp paired-end reads were generated.