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Persistent Identifier
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doi:10.18419/darus-628 |
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Publication Date
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2020-01-30 |
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Title
| Deep enzymology data related to Adam et al.: DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation |
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Subtitle
| Data Set 2 related to enzyme assays with random flank substrate methylation |
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Author
| Jeltsch, Albert (Universität Stuttgart) - ORCID: 0000-0001-6113-9290
Bashtrykov, Pavel (Universität Stuttgart) - ORCID: 0000-0003-3838-2019
Adam, Sabrina (Universität Stuttgart) |
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Point of Contact
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Use email button above to contact.
Jeltsch, Albert (Universität Stuttgart) |
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Description
| Methylation of random flank substrates: Libraries of double stranded DNA substrates with unmethylated or hemimethylated CpG sites in randomized sequence context were methlyated by DNMT1. 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). Sequencing was performed at the Max Planck Genome Centre Cologne. |
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Subject
| Medicine, Health and Life Sciences |
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Keyword
| DNA Methylation http://purl.obolibrary.org/obo/NCIT_C17961 (NCIT)
DNA Methyltransferase http://purl.obolibrary.org/obo/NCIT_C16508 (NCIT)
NGS Bisulfite Sequencing
Enzyme Assay http://purl.obolibrary.org/obo/OMIT_0026837 (OMIT)
DNMT1 http://purl.obolibrary.org/obo/OMIT_0031451 (OMIT) |
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Topic Classification
| Biochemistry (DFGFO) https://github.com/tibonto/dfgfo/201-01 |
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Related Publication
| Sabrina Adam, Hiwot Anteneh, Maximilian Hornisch, Vincent Wagner, Jiuwei Lu, Nicole E. Radde, Pavel Bashtrykov, Jikui Song, Albert Jeltsch (2020) DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation. Nat Commun. 11(1):3723. doi: 10.1038/s41467-020-17531-8 https://doi.org/10.1038/s41467-020-17531-8 |
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Notes
| pmid: 32709850 |
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Funding Information
| DFG: JE 252/36 - 403074082 |
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Depositor
| Jeltsch, Albert |
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Deposit Date
| 2020-01-30 |
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Data Type
| Raw DNA sequences extracted from Fastq NGS files; Bisulfite-seq DNA methylation analysis |
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Related Dataset
| Jeltsch, Albert, 2020, "Deep enzymology data related to Gao et al.: Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms.", DOI: 10.18419/darus-627, DaRUS.; Jeltsch, Albert, 2020, "Deep enzymology data related to Adam et al.: Sequence context-dependent base flipping regulates DNMT1-mediated genome-wide DNA methylation", DOI: 10.18419/darus-629, DaRUS. |
