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Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA

核酸外切酶消化联合选择性化学标记碱基分辨率定位基因组中DNA中5-羟甲基胞嘧啶

Aurélien A. Sérandour Aurélien A. Sérandour
SA Stéphane Avner
GS Gilles Salbert

发布: 2018年03月05日第8卷第5期 DOI: 10.21769/BioProtoc.2747 浏览次数: 7884

评审: Gal HaimovichOmar AkilAnonymous reviewer(s)

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Abstract

This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis.

Keywords: 5-Hydroxymethylcytosine (5-羟甲基胞嘧啶) search Selective chemical labeling (选择性化学标记) search Exonuclease digestion (核酸外切酶消化) search CpG (CpG) search

Background

The methylation of cytosine in genomic DNA can be read by proteins and is mainly translated into gene silencing. Most CpG dinucleotides in the genome are methylated, including those located in gene regulatory regions such as enhancers. However, when required, these CpGs can be demethylated through oxidation of the methyl group by Ten Eleven Translocation (TET) enzymes and replacement by unmethylated cytosines by the base excision repair system. 5-Hydroxymethylcytosine (5hmC) is the first oxidative derivative of 5-methylcytosine, and mapping this modified base in the genome provides information on the regions undergoing active demethylation. Although selective chemical labeling (SCL) allows very specific detection of 5hmC, the resolution of this technique is limited by the size of the DNA fragments, especially when several CpGs are present in the captured DNA. In order to improve resolution, we have introduced a digestion step using exonucleases which trim the DNA molecule up to close proximity of the hydroxymethylated cytosines (Sérandour et al., 2016). Appropriate bioinformatic treatment of the sequencing reads then assigns hydroxymethylation score to the captured CpGs.

Materials and Reagents

  1. Pipette tips (TipOne, STARLAB, catalog numbers: S1161-1800 , S1182-1830 , and S1181-3810 )
  2. 0.65 ml Bioruptor microtubes (Diagenode, catalog number: C30010011 )
  3. 0.5 ml and 2 ml DNA LoBind tubes (Eppendorf, catalog numbers: 0030108035 and 0030108078 )
  4. Micro Bio-Spin 6 column (Bio-Rad Laboratories, catalog number: 7326221 )
  5. 1.5 ml Lobind tubes (Eppendorf, catalog number: 0030108051 )
  6. 2 ml Lobind tubes (Eppendorf, catalog number: 0030108078 )
  7. DNeasy Blood & Tissue Kit (QIAGEN, catalog number: 69504 )
  8. 100-bp DNA marker (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15628019 )
  9. E-gel EX agarose gel 2% (Thermo Fisher Scientific, InvitrogenTM, catalog number: G401002 )
  10. β-Glucosyltransferase (β-GT) and associated reaction buffer (New England Biolabs, catalog number: M0357S )
  11. DBCO-PEG4-Biotin (Sigma-Aldrich, catalog number: 760749 )
  12. UDP-6-N3-Glc (Active Motif, catalog number: 55020 )
  13. DMSO (Sigma-Aldrich, catalog number: D8418 )
  14. QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
  15. Dynabeads M-280 streptavidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11205D )
  16. NEBuffer 2 (New England Biolabs, catalog number: B7002S )
  17. 10x NEBuffer 4 (New England Biolabs, catalog number: M0357S )
  18. ATP (10 mM) (New England Biolabs, catalog number: P0756S )
  19. dNTP solution mix (New England Biolabs, catalog number: N0447S )
  20. T4 DNA polymerase (New England Biolabs, catalog number: M0203S )
  21. DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs, catalog number: M0210S )
  22. T4 PolyNucleotide Kinase (New England Biolabs, catalog number: M0201S )
  23. T4 DNA ligase high concentration (New England Biolabs, catalog number: M0202T )
  24. Nuclease-free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
  25. SCL-exo P7 adapter: annealing of 2 oligonucleotides (5’ Phos = phosphorylated 5’ end):
    P7 exo-adapter reverse: 5’ Phos-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-OH 3’
    P7 exo-adapter forward: 5’ OH-GATCGGAAGAGCACACGTCT-OH 3’
  26. Phi29 polymerase (New England Biolabs, catalog number: M0269S )
  27. Lambda exonuclease (New England Biolabs, catalog number: M0262S )
  28. RecJf exonuclease (New England Biolabs, catalog number: M0264S )
  29. Glycogen (5 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9510 )
  30. Sodium chloride (NaCl) (Acros Organics, catalog number: AC207790050 )
  31. EtOH (100%) (VWR, catalog number: 20821.310 )
  32. SCL-exo P7 primer:
    5’ OH-GACTGGAGTTCAGACGTGTGCT-OH 3’
  33. Agencourt AMPure XP (Beckman Coulter, catalog number: A63880 )
  34. Qiagen MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
  35. SCL-exo P5 adapter: annealing of 2 oligonucleotides:
    P5 exo-adapter reverse: 5’ OH-AGATCGGAAGAGCG-OH 3’
    P5 exo-adapter forward: 5’ OH-TACACTCTTTCCCTACACGACGCTCTTCCGATCT-OH 3’
  36. NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs, catalog number: M0541S )
  37. SCL-exo universal P5 PCR primer (* = Phosphorothioates S-linkage):
    5’ OH-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG*A-OH 3’
  38. SCL-exo index P7 PCR primer (* = Phosphorothioates S-linkage) (index sequences come from TruSeq LT):
    Index 2:
    5’ OH-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 4:
    5’ OH-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 5:
    5’ OH-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 6:
    5’ OH-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 7:
    5’ OH-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 12:
    5’ OH-CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 13:
    5’ OH-CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 14:
    5’ OH-CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 15:
    5’ OH-CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 16:
    5’ OH-CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 18:
    5’ OH-CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Index 19:
    5’ OH-CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCAGACGTGTGC*T-OH 3’
    Notes (concerning the oligonucleotides):
    1. All oligonucleotides were produced by Sigma-Aldrich, purified by HLPC and resuspended in water at 100 μM final.
    2. The SCL-exo P7 adapter and the SCL-exo P5 adapter were obtained by mixing pairs of complementary oligonucleotides in 4 volumes of Annealing buffer (see Recipes) and annealed by heating for 5 min at 95 °C then let cool down slowly to room temperature.
    3. The oligonucleotides designed for SCL-exo were adapted from the P5 and P7 oligonucleotide sequences from Illumina ©2007-2012 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers are authorised for use with Illumina instruments and products only. All other uses are strictly prohibited.
  39. Agilent High Sensitivity DNA Kit (Agilent Technologies, catalog number: 5067-4626 )
  40. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
  41. EDTA (500 mM, pH 8.0) (AppliChem, catalog number: A4892,0500 )
  42. HEPES (1 M) (GibcoTM, catalog number: 15630056 )
  43. Na deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  44. NP-40, IGEPAL® CA-630 (Sigma-Aldrich, catalog number: I8896 )
  45. Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: 62476 )
  46. Magnesium chloride hexahydrate (MgCl2·6H2O) (Merck, catalog number: 442611 )
  47. Ammonium sulfate ((NH4)2SO4) (Merck, catalog number: 101217 )
  48. DTT (Sigma-Aldrich, catalog number: D9779 )
  49. Tris (MP Biomedicals, catalog number: 04819638 )
  50. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H9892 )
  51. Formamide for molecular biology (Sigma-Aldrich, catalog number: F9037 )
  52. 1x PBS (Fisher Scientific, catalog number: BP399 )
  53. Annealing buffer (see Recipes)
  54. RIPA buffer (see Recipes)
  55. Nick Repair buffer low DTT 10x (see Recipes)
  56. TE buffer (see Recipes)
  57. Elution buffer (see Recipes)
  58. Binding & Washing (B&W) buffer (see Recipes)

Equipment

  1. PIPETMAN ClassicTM Pipets (Gilson, catalog numbers: F123600 , F144801 , F123602 and F123615 )
  2. Bioruptor Pico with water cooler (Diagenode, catalog numbers: B01060001 and B02010003 )
  3. E-gel Power Snap Electrophoresis Device (Thermo Fisher Scientific, InvitrogenTM, catalog number: G8100 )
  4. Qubit 3 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q33216 )
  5. Refrigerated centrifuge (Eppendorf, model: 5424 R )
  6. Thermocycler ProFlex PCR system (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4484073 )
  7. ThermoMixer C and Eppendorf ThermoTop (Eppendorf, catalog numbers: 5382000015 and 5308000003 )
  8. DynaMag-2 Magnet (Thermo Fisher Scientific, catalog number: 12321D )
  9. Speed-Vac Savant (Thermo Fisher Scientific, catalog number: DNA120-115 )
  10. 2100 Bioanalyzer Instrument (Agilent Technologies, model: 2100, catalog number: G2939BA )
  11. Mini centrifuge (Bio-Rad Laboratories, catalog number: 1660603 )

Procedure

English
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文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Sérandour, A. A., Avner, S. and Salbert, G. (2018). Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA. Bio-protocol 8(5): e2747. DOI: 10.21769/BioProtoc.2747.

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分类

系统生物学 > 表观基因组学 > 5-羟甲基胞嘧啶

分子生物学 > DNA > DNA 修饰

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