Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells
Abstract
:1. Introduction
2. Results
2.1. Analysis of DNA Methylation at FspEI Sites during ESC Differentiation
2.2. Distribution of DNA Methylation Changes in ESCs during Differentiation
2.3. DNA Methylation in LSD1-Bound Enhancers is Affected by LSD1 Inhibition
2.4. Sparsely Methylated Enhancers Are Strongly Affected by LSD1 Inhibition
2.5. Enhancers with Intermediate CpG Density Are Sensitive to LSD1 Inhibition
3. Discussion
4. Materials and Method
4.1. ESC Culture and Differentiation Method
4.2. MethylRAD Library Preparation and Sequencing
4.3. Alignment and Quality Control
4.4. Methylation Site Identification and Quantification
4.5. Comparing MethylRAD Data to RRBS
4.6. Annotation to LSD1-Bound Enhancers
4.7. Comparison of Methylation Levels between Samples
4.8. Calculating CpG Content
4.9. Pathway Analysis
4.10. DNA Methylation-Dependent qPCR Assay (MD-qPCR)
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Samples | Total Reads | Clean Data | Reads with FspEI Site | Reads Mapped | % Uniquely Mapped Reads |
---|---|---|---|---|---|
UD | 128,245,179 | 128,244,535 | 67,257,685 | 50,959,931 | 76% |
Untreated | 191,444,768 | 191,547,179 | 103,231,509 | 73,712,891 | 71% |
Treated | 184,231,969 | 184,230,068 | 101,304,461 | 67,235,311 | 66% |
CCGG | CCWGG | |||
---|---|---|---|---|
Sample | # Sites | Average Depth | # Sites | Average Depth |
UD | 870,345 | 36.29 | 538,222 | 15.96 |
Untreated | 882,955 | 41.48 | 1,313,633 | 15.24 |
Treated | 892,366 | 28.84 | 1,162,815 | 14.00 |
Sample | Sites Intersecting with RRBS (>5 Reads per Site) | Percent MethylRAD Overlap | Percent RRBS Overlap |
---|---|---|---|
UD | 126625 | 9% | 20% |
Untreated | 130904 | 9% | 21% |
Treated | 138419 | 7% | 22% |
All MethylRAD | 164345 | 5% | 26% |
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Saha, D.; Norvil, A.B.; Lanman, N.A.; Gowher, H. Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells. Epigenomes 2020, 4, 24. https://0-doi-org.brum.beds.ac.uk/10.3390/epigenomes4040024
Saha D, Norvil AB, Lanman NA, Gowher H. Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells. Epigenomes. 2020; 4(4):24. https://0-doi-org.brum.beds.ac.uk/10.3390/epigenomes4040024
Chicago/Turabian StyleSaha, Debapriya, Allison B. Norvil, Nadia A. Lanman, and Humaira Gowher. 2020. "Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells" Epigenomes 4, no. 4: 24. https://0-doi-org.brum.beds.ac.uk/10.3390/epigenomes4040024