Deep enzymology data related to Adam et al.: DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation (doi:10.18419/darus-628)

View:

Part 1: Document Description
Part 2: Study Description
Part 5: Other Study-Related Materials
Entire Codebook

Deep enzymology data related to Adam et al.: DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation

doi:10.18419/darus-628

DaRUS

2020-01-30

1

Jeltsch, Albert; Bashtrykov, Pavel; Adam, Sabrina, 2020, "Deep enzymology data related to Adam et al.: DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation", https://doi.org/10.18419/darus-628, DaRUS, V1

Deep enzymology data related to Adam et al.: DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation

Data Set 2 related to enzyme assays with random flank substrate methylation

doi:10.18419/darus-628

Jeltsch, Albert (Universität Stuttgart)

Bashtrykov, Pavel (Universität Stuttgart)

Adam, Sabrina (Universität Stuttgart)

JE 252/36 - 403074082

DaRUS

Jeltsch, Albert

Jeltsch, Albert

2020-01-30

https://doi.org/10.18419/darus-628

Medicine, Health and Life Sciences, DNA Methylation, DNA Methyltransferase, NGS Bisulfite Sequencing, Enzyme Assay, DNMT1

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.

Raw DNA sequences extracted from Fastq NGS files; Bisulfite-seq DNA methylation analysis

<a href="https://pubmed.ncbi.nlm.nih.gov/32709850/">pmid: 32709850</a>

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.", <a href="https://doi.org/10.18419/darus-627">DOI: 10.18419/darus-627</a>, DaRUS.

Jeltsch, Albert, 2020, "Deep enzymology data related to Adam et al.: Sequence context-dependent base flipping regulates DNMT1-mediated genome-wide DNA methylation", <a href="https://doi.org/10.18419/darus-629">DOI: 10.18419/darus-629</a>, DaRUS.

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.

10.1038/s41467-020-17531-8

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.

Label:

hemimethylated Repeat 1.txt

text/plain

Label:

hemimethylated Repeat 2.txt

text/plain

Label:

hemimethylated without enzyme.txt

text/plain

Label:

readme_random_flank_substrate.pdf

Text:

Additional information

application/pdf

Label:

unmethylated with enzyme.txt

text/plain

Label:

unmethylated without enzyme.txt

text/plain