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Reactions were stopped by shock freezing in liquid nitrogen, then treated with proteinase K for 2 hours. Afterwards, the DNA was digested with the BsaI-HFv2 enzyme and a hairpin was ligated using T4 DNA ligase (NEB). The DNA was bisulfite converted using EZ DNA Methylation-Lightning kit (ZYMO RESEARCH) according to the manufacturer protocol, purified and eluted with 10 µL ddH2O. Libraries for Illumina Next Generation Sequencing (NGS) were produced with the two-step PCR approach. In the first PCR, 2 µL of bisulfite-converted DNA were amplified with the HotStartTaq DNA Polymerase (QIAGEN) and primers containing internal barcodes using following conditions: 15 min at 95 °C, 10 cycles of 30 sec at 94 °C, 30 sec at 50 °C, 1 min and 30 sec at 72 °C, and final 5 min at 72 °C; using a mixture containing 1x PCR Buffer, 1x Q-Solution, 0.2 mM dNTPs, 0.05 U/µL HotStartTaq DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. In the second PCR, 1 µL of obtained products were amplified by Phusion Polymerase (Thermo) with another set of primers to introduce adapters and indices needed for NGS (30 sec at 98 °C, 10 cycles - 10 sec at 98 °C, 40 sec at 72 °C, and 5 min at 72 °C). PCRII was carried out in 1x Phusion HF Buffer, 0.2 mM dNTPs, 0.02 U/µL Phusion HF DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. Obtained libraries were pooled in equimolar amounts and purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel), followed by a second purification step of gel extraction and size exclusion with AMPure XP magnetic beads (Beckman Coulter)."},"subject":"Medicine, Health and Life Sciences","citation:keyword":[{"citation:keywordValue":"DNA Methylation","citation:keywordVocabulary":"WikiData","citation:keywordVocabularyURI":"https://www.wikidata.org/wiki/Q874745"},{"citation:keywordValue":"DNA Methyltransferase","citation:keywordVocabulary":"NCIT","citation:keywordVocabularyURI":"http://purl.obolibrary.org/obo/NCIT_C16508"},{"citation:keywordValue":"NGS Bisulfite Sequencing"},{"citation:keywordValue":"Enzyme Assay","citation:keywordVocabulary":"WikiData","citation:keywordVocabularyURI":"https://www.wikidata.org/wiki/Q5381543"},{"citation:keywordValue":"DNMT3B","citation:keywordVocabulary":"WikiData","citation:keywordVocabularyURI":"https://www.wikidata.org/wiki/Q15313456"},{"citation:keywordValue":"Enzyme Activity","citation:keywordVocabulary":"WikiData","citation:keywordVocabularyURI":"https://www.wikidata.org/wiki/Q22035510"}],"publication":{"publicationCitation":"Michael Dukatz, Sabrina Adam, Mahamaya Biswal, Jikui Song, Pavel Bashtrykov, & Albert Jeltsch: Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase. 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