通过 dA6m DNA 免疫沉淀法识别基因组DNA中的甲基化脱氧腺苷位点

Magdalena  J. Koziol; Charles  R. Bradshaw; George  E. Allen; Ana S. H. Costa; Christian  Frezza

doi:10.21769/BioProtoc.1990

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Peer-reviewed

Identification of Methylated Deoxyadenosines in Genomic DNA by dA^6m DNA Immunoprecipitation

通过 dA6m DNA 免疫沉淀法识别基因组DNA中的甲基化脱氧腺苷位点

Magdalena J. Koziol email

CB Charles R. Bradshaw

GA George E. Allen

Ana S. H. Costa

Christian Frezza

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1990 浏览次数: 10271

评审: Gal HaimovichZhen ShiBenoit Chassaing

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Abstract

dA^6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N⁶-methyldeoxyadenosine (dA^6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA^6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA^6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA^6m is pulled down with an antibody that recognizes dA^6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA^6m are eliminated, and the dA^6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses.

Background

This protocol was developed in order to identify regions in the genome that contain dA^6m. It can be used to detect dA^6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA^6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA^6m in eukaryotic DNA.

Materials and Reagents

Microcentrifuge tubes, 1.7 ml (Coring, Axygen^®, catalog number: MCT-175-C )
1.5 ml Bioruptor Pico microtubes with caps (Diagenode, catalog number: C30010016 )
Optional: D1000 tape and reagents (Agilent Technologies, catalog numbers: 5067-5582 ; 5067-5583 )
DNeasy Blood & Tissue Kit (QIAGEN, catalog number: 69506 )
Phosphate-buffered saline (PBS)
CutSmart^® buffer (New England Biolabs, catalog number: B7204S )
RNase (DNase-free) (Roche Diagnostics, catalog number: 11119915001 )
Water (double deionized water that has been autoclaved)
Note: All water used in this protocol refers to double deionized water, which has been subsequently autoclaved. As an alternative, purchased DNAse free water could be used.
Qubit^® dsDNA HS Assay Kit (Thermo Fisher Scientific, Invitrogen^TM, catalog number: Q32854 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3059 )
dA^6m antibodies (Synaptic Systems, catalog numbers: 202011 / 202111 / 202003 )
Dynabeads^® protein A (Thermo Fisher Scientific, Novex^TM, catalog number: 10008D )
Glycogen (Roche Diagnostics, catalog number: 10901393001 )
NaOAc (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10010500 )
Isopropanol (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10315720 )
Ethanol absolute (VWR, catalog number: 20821330 )
TruSeq Nano DNA LT Library Preparation Kit (set A or B) (Illumina, catalog number: FC-121-4001 or FC-121-4002 )
Tris-HCl (Melford Laboratories, catalog number: B2005 )
Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10428420 )
Igepal CA-630 (Sigma-Aldrich, catalog number: I8896 )
N⁶-methyladenosine 5’-monophosphate sodium salt (Sigma-Aldrich, catalog number: M2780 )
EDTA (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10213570 )
Dry ice
5x DIP IP buffer (see Recipes)
1x DIP IP buffer (see Recipes)
DIP elution buffer (see Recipes)

Equipment

Incubator (Grant, dry block heating system)
Rotating wheel (Bibby Scientific, Stuart, model: rotator SB3 )
Thermomixer (Eppendorf, model: Thermomixer compact )
Qubit fluorometer (Thermo Fisher Scientific, Invitrogen^TM, catalog number: Q32857 )
Note: This product has been discontinued.
Water bath sonicator (Diagenode, Bioruptor Pico, catalog number: B01060001 )
Microcentrifuge (Eppendorf, model: Centrifuge 5424 )
Vortex (Scientific Industries, model: Vortex-Genie 2 )
Magnet (Thermo Fisher Scientific, DynaMag^TM, catalog number: 12321D )
Optional: 2200 Tapestation (Agilent Technologies, catalog number: G2965A )

Procedure

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如何引用

Koziol, M. J., Bradshaw, C. R., Allen, G. E., Costa, A. S. H. and Frezza, C. (2016). Identification of Methylated Deoxyadenosines in Genomic DNA by dA^6m DNA Immunoprecipitation. Bio-protocol 6(21): e1990. DOI: 10.21769/BioProtoc.1990.