Genomic DNA Extraction and Bisulfite Sequencing from Chicken Spermatozoa

Genomic DNA was extracted and purified from chicken spermatozoa using an AllPrep DNA/RNA Micro Kit (QIAGEN) following the manufacturer’s instructions as previously described by Kito, et al.^{68 (link)}. Sodium bisulfite conversion was performed using 1 μl of genomic DNA (1 μg/μl), 19 μl of RNase-free water, 85 μl of Bisulfite mix, and 35 μl of DNA protect buffer with a Master Cycler Gradient (Eppendorf, Hamburg, Germany) following to the manufacturer’s instructions of the EpiTech Bisulfite Kit (QIAGEN). Bisulfite-sequencing PCR (BSP) amplification was performed using 1 μl of purified bisulfite-converted DNA (50 ng/μl), 10 μl of 2× Prime Taq Premix, 1 μl of upstream primer (5 pmol/μl), 1 μl of downstream primer (5 pmol/μl), and 7 μl of DEPC-treated water. Primers for BSP were designed using the MethPrimer (http://www.urogene.org/methprimer/) (see Supplementary Table S4). BSP amplification products were purified using the MiniBest Agarose Gel DNA Extraction Kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) following the manufacturer’s instructions.