RNA/DNA 亚硫酸氢盐转化实验-Bisulfite assay
一、RNA bisulfite
1、原理示意图
2、RNA bisulfite方法总结
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Li Q, Li X, Tang H, Jiang B, Dou Y, Gorospe M, Wang W. NSUN2-Mediated m5C Methylation and METTL3/METTL14-Mediated m6A Methylation Cooperatively Enhance p21 Translation. J Cell Biochem. 2017 Sep;118(9):2587-2598.
- a) 70°C for 5 min and 60°C for 60min, repeating for 3–5 cycles
- b) Desulfonated by 1M Tris (pH 9.0, 1/1, V/V) at 37°C for 1 h
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Khoddami V, Yerra A, Mosbruger TL, Fleming AM, Burrows CJ, Cairns BR. Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution. Proc Natl Acad Sci U S A. 2019 Apr 2;116(14):6784-6789.
- a) 50 °C 时间未写
- b) desulfonate by 2 M Tris buffer, pH 9.0 for 2 h at 37 °C.
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Selmi T, Hussain S, Dietmann S, Heiß M, Borland K, Flad S, Carter JM, Dennison R, Huang YL, Kellner S, Bornelöv S, Frye M. Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6. Nucleic Acids Res. 2021 Jan 25;49(2):1006-1022.
- a) 1h at 60°C
- b) Desulphonated by adding an equal volume of 1 M Tris (pH 9.0) for 1 h at 37°C
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Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T. Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 2012 Jun;40(11):5023-33.
- a) 75°C for 4 h
- b) desulfonated by adding an equal volume of 1 M Tris (pH 9.0) 1 h at 75°C
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Yang X, Yang Y, Sun BF, Chen YS, Xu JW, Lai WY, Li A, Wang X, Bhattarai DP, Xiao W, Sun HY, Zhu Q, Ma HL, Adhikari S, Sun M, Hao YJ, Zhang B, Huang CM, Huang N, Jiang GB, Zhao YL, Wang HL, Sun YP, Yang YG. 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader. Cell Res. 2017 May;27(5):606-625.
- a) 75°C for 4 h.
- b) desulfonated 1 M Tris (pH 9.0) for 1 h at 75°C
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Amort T, Rieder D, Wille A, Khokhlova-Cubberley D, Riml C, Trixl L, Jia XY, Micura R, Lusser A. Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain. Genome Biol. 2017 Jan 5;18(1):1.
- a) 70°C for 5 minutes
- b) 54°C for 45 minutes
- c) desulfonated (20°C – 30°C) for 30 minutes.
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David R, Burgess A, Parker B, Li J, Pulsford K, Sibbritt T, Preiss T, Searle IR. Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs. Plant Cell. 2017 Mar;29(3):445-460.
- a) 75°C for 4 h.
- b) desulfonated 1 M Tris (pH 9.0) for 1 h at 75°C
二、DNA bisulfite
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Miura F, Enomoto Y, Dairiki R, Ito T. Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 2012 Sep 1;40(17):e136.
- a) 99°C for 6 min and incubated at 65°C for 90 min.
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Xue JH, Chen GD, Hao F, Chen H, Fang Z, Chen FF, Pang B, Yang QL, Wei X, Fan QQ, Xin C, Zhao J, Deng X, Wang BA, Zhang XJ, Chu Y, Tang H, Yin H, Ma W, Chen L, Ding J, Weinhold E, Kohli RM, Liu W, Zhu ZJ, Huang K, Tang H, Xu GL. A vitamin-C-derived DNA modification catalysed by an algal TET homologue. Nature. 2019 May;569(7757):581-585.
- a) 98°C for 8 minutes
- b) 64°C for 3.5 hours
- c) 4°C storage for up to 20 hours
- d) desulfonated at temperature (20-30°C) for 15-20 minutes.
EZ DNA Methylation-Direct Kit (Zymo Research)
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Wang L, Zhang J, Duan J, Gao X, Zhu W, Lu X, Yang L, Zhang J, Li G, Ci W, Li W, Zhou Q, Aluru N, Tang F, He C, Huang X, Liu J. Programming and inheritance of parental DNA methylomes in mammals. Cell. 2014 May 8;157(4):979-991.
- a) 64°C 2 h
- b) desulfonated at temperature (20-30°C) for 15-20 minutes.
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Guo F, Li X, Liang D, Li T, Zhu P, Guo H, Wu X, Wen L, Gu TP, Hu B, Walsh CP, Li J, Tang F, Xu GL. Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell. 2014 Oct 2;15(4):447-459.
a) 98°C for 8 minutes
b) 64°C for 3.5 hours
c) 4°C storage for up to 20 hours room
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d) desulfonated at temperature (20-30°C) for 15-20 minutes.
EZ DNA Methylation-Direct Kit (Zymo Research)
