Epitect kit

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.

DNA methylation levels of the 5′ MUPCDH promoter region were analyzed in DNA from 4 cell lines and FFPE derived DNA from 24 cancer samples, 12 adenomas, and 10 normal samples (obtained from swabs of normal colon epithelium). The analysis involved the region located at chr11:624,959-625,053 (grch37/hg19 genome build) that contains 9 particular CpG sites within 95 bp DNA sequence.
The FFPE tissue samples were manually macrodissected to reduce the content of nonneoplastic cells. DNA was isolated using the QIAamp DNA Mini Kit (Qiagen) and bisulfite was converted using EpiTect kit (Qiagen), according to manufacturer's recommendations. The PCR reaction consisted of a 30 μL mixture containing 1x PCR buffer, 2 mM MgCl₂, 0.25 mM dNTPs, 0.2 μM of each primer, and 0.5 U of FastStart DNA Polymerase (Roche Applied Science) with the following cycling conditions: 94°C for 3 min, followed by 40 cycles of 30 s at 94°C, 30 s at the annealing temperature of 54°C, and 30 s at 72°C with final incubation for 7 min at 72°C. Primers' sequences are shown in Table 2.
The 188 bp long PCR products were analyzed by the PyroMark Q24 System (Qiagen), according to the published protocol [17 (link)]. For each sample, the mean methylation level of CpGs located within the analyzed region was used for statistical evaluation.