Instrument: Illumina HiSeq 2500
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: Reduced Representation
Layout: PAIRED
Construction protocol: Testes and cauda epidymides from mature male SDs were dissected, washed with PBS and placed in 35mm petri dishes containing HBSS supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO4•7H2O, 1.3 mM CaCl2•2H2O, 6.6 mM sodium pyruvate, 0.05% lactate, 2 mM GlutaMAXT Supplement (Gibco®) and 0.5% BSA. Released sperm-containing supernatant was moved to 5 ml collection tubes and let stand for 10 min at 37℃ for swimming up. Supernatant was further filtered through 40 μm nylon mesh and laid on top of a three-layer gradient of 80%, 60% and 30% Percol, centrifuged for 10 min at 1,000 g at room temperature. Sperms were collected from 60% layer of the gradient, washed with the incubation buffer and cryopreserved. DNA was extracted from purified sperm by lysis with ,phenol-chloroform extraction followed by isopropanol precipitation and then preserved in TE buffer. For library preparation, 1 mg genomic DNA was digested by MspI to generate short fragments that contain CpG dinucleotides at the ends. Then digested DNA was end-repaired, A-tailed and ligated to methylated Illumina adapters following the user manual of TruseqTM DNA Sample Prep Kit (Illumina, USA) Then, the CpG-rich DNA fragments (160-340 bp) are size selected, subjected to bisulfite conversion by MethylCode™ Bisulfite Conversion Kit (Life Technologies, USA) , and PCR amplified by ZymoTaqTM PreMix (Zymo, USA). Barcodes are indicated in the file names of each raw .fastq.gz file.