Amplicon TSDR Methylation Analysis

DNA was prepared using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer´s protocol. Up to 200 ng genomic DNA or all DNA obtained from pellets containing min. 400 cells was bisulfite-converted using OPTI-Bisulfite (36) or EZ-DNA methylation Gold kit (Zymo, Irvine, USA). Subsequently PCRs were performed (human: 2-5 µl of bisulfite-treated DNA, 1xBD buffer (Solis Biodyne, Tartu, Estonia), 0.25 mM of each dNTP, 0.25 mM MglCl₂, 2.5U HotFirePol (Solis Biodyne) - murine: 10 µl bisulfite-treated DNA, 80 mM Tris-HCL, 20 mM (NH₄)₂SO₄, 0.2% Tween-20, 2.5 mM MgCl₂, 0.2 mM of each dNTP, 2.5U HotFirePol) using 0.5 pmol of primers (human_F:5’TTGTTTGGGGGTAGAGGATTTAG-3’, human_R:5’CCTAATATTATACTATTTAAAAACCCC-3’) (murine: 5 pmol of each primer murine_F: 5´-GGGTTTTTTTGGTATTTAAGAAAGAT-3´, murine_R: 5´-AAATCTACATCTAAACCCTATTATCACA-3) with Illumina compatible universal adaptor sequences attached at the 5´-end. PCRs were performed in a thermocycler starting with 15 min 95°C followed by 45 cycles 95°C 1 min, 56°C 2 min (murine 52°C), 72°C 1 min and a 10 min final extension at 72°C. Amplicons were purified with MagSi-NGS Prep Plus beads (Steinbrenner, Wiesenbach, Germany), diluted, pooled and sequenced on the MiSeq platform (v3 chemistry: 2x300 bp paired-end, Illumina, San Diego, USA) following the manufacturer’s instructions aiming at 10,000 reads per amplicon. Reads were aligned and evaluated using the semi-automated tool BiQ-Analyzer HT (37). Read pattern maps were generated in R using in-house scripts.