Parallel Sperm Methylome Analysis via RRBS

Parallel samples from four of the eight individuals belonging to D1-2°C, D1-2°C-Cryo, D4-2°C, and D4-8°C groups were considered for the construction of RRBS libraries using a gel-free multiplexed technique (Boyle et al., 2012 (link)), previously optimized to study sperm DNA methylation in boar (Khezri et al., 2019 (link)) and bull (Khezri et al., 2020 (link)). In brief, 100 ng genomic DNA isolated from milt samples was digested overnight at 37°C using MspI and Taqα1 enzymes (New England Biolabs). Fragmented DNA was subjected to Gap filling, A-tailing, and size selection (300–500 bp). Size selected DNA samples were adapter-ligated using NEXTflex™ Bisulfite-Seq barcodes (Bio Scientific Corporation) and were bisulfite converted using EpiTect kit (QIAGEN, Germany) following the manufacturer’s protocol. The product was cleaned up according to recommendations in the QIAGEN EpiTect kit (Gu et al., 2011 (link)) and PCR amplified. The PCR product was further enriched by adding 1× SPRI AMPure XP beads. The DNA concentration of eluted and cleaned RRBS libraries was measured using the PicoGreen dsDNA absorbance method. The prepared libraries were sent to the Norwegian Sequencing Centre where sequencing was performed by Illumina HiSeq 4000 in paired-end (2 × 150 bp) mode.