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Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m 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   

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Abstract

dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). 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 dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m 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 dA6m. It can be used to detect dA6m 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 dA6m 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 dA6m in eukaryotic DNA.

Materials and Reagents

  1. Microcentrifuge tubes, 1.7 ml (Coring, Axygen®, catalog number: MCT-175-C )
  2. 1.5 ml Bioruptor Pico microtubes with caps (Diagenode, catalog number: C30010016 )
  3. Optional: D1000 tape and reagents (Agilent Technologies, catalog numbers: 5067-5582 ; 5067-5583 )
  4. DNeasy Blood & Tissue Kit (QIAGEN, catalog number: 69506 )
  5. Phosphate-buffered saline (PBS)
  6. CutSmart® buffer (New England Biolabs, catalog number: B7204S )
  7. RNase (DNase-free) (Roche Diagnostics, catalog number: 11119915001 )
  8. 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.
  9. Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
  10. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3059 )
  11. dA6m antibodies (Synaptic Systems, catalog numbers: 202011 / 202111 / 202003 )
  12. Dynabeads® protein A (Thermo Fisher Scientific, NovexTM, catalog number: 10008D )
  13. Glycogen (Roche Diagnostics, catalog number: 10901393001 )
  14. NaOAc (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10010500 )
  15. Isopropanol (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10315720 )
  16. Ethanol absolute (VWR, catalog number: 20821330 )
  17. TruSeq Nano DNA LT Library Preparation Kit (set A or B) (Illumina, catalog number: FC-121-4001 or FC-121-4002 )
  18. Tris-HCl (Melford Laboratories, catalog number: B2005 )
  19. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10428420 )
  20. Igepal CA-630 (Sigma-Aldrich, catalog number: I8896 )
  21. N6-methyladenosine 5’-monophosphate sodium salt (Sigma-Aldrich, catalog number: M2780 )
  22. EDTA (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10213570 )
  23. Dry ice
  24. 5x DIP IP buffer (see Recipes)
  25. 1x DIP IP buffer (see Recipes)
  26. DIP elution buffer (see Recipes)

Equipment

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

Procedure

The procedure can be divided into three parts: ‘A’ isolation of genomic DNA, ‘B’ sonication and ‘C’ DNA immunoprecipitation of dA6m. An outline of the procedure is illustrated in Figure 1. We encourage carrying out all experiments with at least two biological replicates, and with at least 2 different dA6m antibodies. As a further control, one could carry out the DNA immunoprecipitation procedure on E. coli bacterial DNA, as described in the original work (Koziol et al., 2015). Wild type bacteria could be used as a positive control, as their dA6m sites are well known. As a negative control, bacteria deficient in the Dam dA6m methylase can be used. Such bacteria no longer have deoxyadenosine methylation within the sequence GATC, and overall have a reduced number of dA6m sites. The success of the DNA immunoprecipitation of dA6m can then be accessed by observing a significant difference between both bacterial genomes in their dA6m signal, specifically by the reduction in dA6m within GATC sequences (Koziol et al, 2015).


Figure 1. Illustration of dA6m DNA immunoprecipitation. Genomic DNA is isolated and any RNAs are eliminated by RNase treatment. Next, the DNA is fragmented and enriched by DNA immunoprecipitation using an antibody against dA6m. The enriched fraction can then be analyzed by subsequent deep sequencing.

  1. Genomic DNA isolation
    1. Extract genomic DNA using the DNeasy Blood & Tissue Kit, following the manufacturer’s guidelines, but with the exception: elute the DNA with 177 μl of PBS, instead of AE buffer (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0). Next, add again 177 μl of PBS onto the column, and elute. Collect both fractions in the same tube, making a total of 354 μl.
      Note: Any method to extract genomic DNA can be used in this step, as long as at least 5 μg of DNA is obtained. If another method of genomic DNA extraction is used, ensure you dilute your genomic DNA so that the final volume is 354 μl. In this volume, the concentration of DNA should be at least 14 ng/μl.
    2. Add 40 μl of CutSmart buffer.
    3. Add 6 μl of RNase DNase-free.
    4. Incubate at 37 °C overnight.
      Note: Incubate for at least 6 h to ensure a good degradation of RNA. RNAs contain methylated adenosines, which the antibody can also recognize. Hence, the presence of RNA might inhibit recovery of methylated deoxyadenosines from DNA making removal important.
    5. Split the sample into two, and clean up the genomic DNA: Use the DNeasy Blood & Tissue Kit, following the manufacturer’s protocol ‘DNeasy Blood & Tissue Kit’, eluting the DNA once with 177 μl of PBS, instead of AE buffer.
      Note: This column cleanup step helps to better eliminate the RNAs from the sample. The split into two columns is required as otherwise the volumes that required to be loaded exceed the column capacity.
    6. Combine the two corresponding samples.
    7. Add 40 μl of CutSmart buffer.
    8. Add 2 μl of RNase DNase-free.
    9. Incubate at 37 °C for at least 2 h.
      Note: This additional RNase step ensures that most of the RNA is degraded.
    10. Divide the sample in two, and clean up the genomic DNA: Use the DNeasy Blood & Tissue Kit, following the manufacturer’s guidelines, eluting the DNA once with 200 μl of water, instead of AE buffer.
      Note: The split into two columns is required as otherwise the volumes that required to be loaded exceed the column capacity.
    11. Combine the two corresponding samples.
    12. Quantify genomic DNA content using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit, by following the manufacturer’s guidelines.
      Note: We recommend not to use Nanodrop for quantification, as this does not result in reliable quantification. At this point, the protocol can be stopped and the DNA can be frozen. We recommend storage at -20 °C or colder.

  2. Sonication of genomic DNA
    1. Take 5 μg of genomic DNA and transfer into a 1.5 ml Bioruptor Pico microtube.
    2. Adjust the volume to 300 μl with water.
    3. Sonicate in Diagenode Bioruptor Pico for 35 cycles, 30 sec on/30 sec off, at 4 °C. A representative example is shown in Figure 2.
      Note: Sonication time needs to be optimized depending on the machine and genomic DNA used. We aim for a DNA fragment size that peaks at about 150-300 base pairs.
    4. Verify fragmentation of genomic DNA with the Tapestation, using the D1000 tape and reagents, following the manufacturer’s protocol.
      Note: Alternatively to the Tapestation, genomic DNA can be run on an agarose gel to verify the fragment size following sonication.

  3. dA6m DNA immunoprecipitation
    1. Take aside 300 ng of sonicated genomic DNA for each sample. This will be the input control for deep sequencing and will not be used for the DNA immunoprecipitation procedure described below. Store at -20 °C until needed.
    2. Take 3 μg of the sonicated genomic DNA, and add to a 1.7 ml microcentrifuge tube.
    3. Place the tube on ice.
    4. On ice, add 200 μl of 5x DIP IP buffer.
    5. On ice, add 200 μl of 30 μg/μl BSA stock solution.
    6. On ice, adjust the total volume to 1 ml with water.
    7. Vortex briefly, about 2 sec, and place on ice.
    8. Add 5 μl of 0.5 μg/μl dA6m antibody stock.
    9. Immediately mix gently by turning the tube and place the tube back on ice.
    10. Mix magnetic protein A beads by vortexing, so that all beads are resuspended.
    11. Take 100 μl of mixed magnetic protein A beads per sample, and add to a new 1.7 ml microcentrifuge tube.
    12. Place on magnet and wait for 1 min for magnetic beads to stick to the side of tube.
    13. Remove the supernatant from the beads.
    14. Remove beads from magnet.
    15. Add 1 ml of 1x DIP IP buffer to the beads and mix gently.
    16. Repeat steps C11-C14 at least two more times.
    17. Place on magnet and wait for 1 min for magnetic beads to stick to the side of tube.
    18. Remove the supernatant from the beads.
    19. Resuspend beads in 105 μl 1x DIP IP buffer.
    20. Add 105 μl of 30 μg/μl BSA stock solution.
    21. Incubate the beads separately on a rotating wheel at 4 °C.
      Note: The beads and samples are incubated in separate tubes.
    22. Incubate the samples separately on a rotating wheel at 4 °C.
    23. After an overnight incubation, add 200 μl of the bead suspension to each sample.
    24. Incubate the sample-bead mixture for at least one more hour on the rotating wheel at 4 °C.
    25. Place sample on magnet and wait for 1min for magnetic beads to stick to the side of tube.
    26. Remove all the supernatant from the beads.
    27. Remove beads from magnet.
    28. Add 1 ml of 1x DIP IP buffer to the beads and mix gently.
    29. Repeat steps C23-C26 at least three more times.
    30. Place sample on magnet and wait for 1 min for magnetic beads to stick to the side of tube.
    31. Remove all supernatant from the beads.
    32. Add 200 μl of the DIP elution buffer.
    33. Vortex.
    34. Place sample in Thermomixer at 42 °C for at least 1 h, at 1,400 rpm.
    35. Spin the samples down.
    36. Place sample on magnet and wait for 1min for magnetic beads to stick to the side of tube.
    37. Transfer the supernatant from the beads into a new 1.7 ml microcentrifuge tube.
      Note: You can discard the beads.
    38. Add 300 μl of water to the recovered supernatant.
    39. Add 2 μl glycogen.
    40. Add 50 μl of 3 M stock of NaOAc.
    41. Add 500 μl isopropanol.
    42. Vortex.
    43. Place on dry ice until frozen.
    44. Centrifuge at maximum speed for 30 min.
      Note: A pellet should have formed, that contains the dA6m enriched DNA.
    45. Remove supernatant.
    46. Wash pellet twice with 1 ml of 70% ethanol.
    47. Remove any liquid.
    48. Let pellet dry for 5 min at room temperature.
    49. Resuspend in desired amount of water.
      Note: We usually resuspend in about 10-50 μl.
    50. Quantify genomic DNA content using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit, by following the manufacturer’s guidelines.
      Note: This step is required to ensure that you have actually obtained genomic DNA with the pulldown. We usually expect to obtain at least 10 ng of genomic DNA.
    51. Use all genomic DNA from step C47 and the corresponding input control from step C1 to make standard deep sequencing libraries.
      Note: Although any standard deep sequencing is possible, we recommend using the TruSeq Nano DNA LT Library Preparation Kit (set A or B) for preparation of libraries suitable for Illumina sequencing with for example HiSeq 2500 or HiSeq 4000.

Data analysis

Any information about data processing and analysis, replicates, independent experiments, reproducibility, including any statistical tests applied, has been provided in the original research paper where this method has been applied (Koziol et al., 2015).

Representative data




Figure 2. Distribution of genomic DNA fragmentation pattern after sonication. 5 μg of genomic DNA was fragmented in a volume of 300 μl for 35 min with the Diagenode sonicator. The size of the genomic DNA was determined by Tapestation analysis, with the D1000 Tapestation reagents. In this sample, the peak of the fragmented DNA was determined to be 245 bp. Lower and upper markers are also shown.

Notes

  1. We have found that this protocol is very robust and reproducible. We were always able to enrich for dA6m and always obtained sufficient DNA for subsequent standard deep sequencing libraries. If the method does not result in enough or no DNA, which we did not experience, repeat the experiment again, maybe with a fresh antibody batch.
  2. We tested 3 different dA6m antibodies for the enrichment of dA6m in the eukaryotic genome. All of which are listed above. We have found that all of them gave reproducible and overlapping results (Koziol et al., 2015). Hence, we believe any of these antibodies will work. However, we encourage confirming any initial results with at least two different dA6m antibodies, to have a higher confidence in the results.

Recipes

Note: Prepare all the solutions fresh from stock solutions before starting and keep on ice.

  1. 5x DIP IP buffer
    Add indicated amount to 7.5 ml water
    0.5 ml of 1 M Tris-HCl (pH 7.4) stock
    1.5 ml of 5 M NaCl stock
    0.5 ml of 10% vol/vol Igepal CA-630 stock
  2. 1x DIP IP buffer
    Mix 10 ml of 5x DIP IP buffer with 40 ml of water
  3. DIP elution buffer
    Add indicated amount for each sample, scale up according to number of samples
    45 μl of 5x DIP IP buffer
    75 μl of 20 mM N6-methyladenosine 5’-monophosphate sodium salt stock
    105 μl of water

Acknowledgments

This protocol was used for the work previously published in Nature Structural and Molecular Biology (Koziol et al., 2015). M.J.K. was supported by the Long-Term Human Frontiers Fellowship (LT000149/2010-L), the Medical Research Council grant (G1001690), and by the Isaac Newton Trust Fellowship (RG76588). The work was sponsored by the Biotechnology and Biological Sciences Research Council grant BB/M022994/1 (M.J.K.). The Gurdon laboratory where this work was carried out is funded by the grant 101050/Z/13/Z from the Wellcome Trust, and is supported by the Gurdon Institute core grants, namely by the Wellcome Trust Core Grant (092096/Z/10/Z) and by the Cancer Research UK Grant (C6946/A14492). C.R.B. and G.E.A. are funded by the Wellcome Trust Core Grant. We thank S. Moss and K. Harnish for their advice and assistance with deep sequencing, who were supported by both Gurdon Institute core grants. We are grateful to Nigel Garrett for his critical comments. This protocol reported here was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013).

References

  1. Dominissini, D., Moshkovitz, M. S., Divon, S. M., Amariglio, N. and Rechavil, G. (2013). Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing. Nature Protocols 8(1): 176-189.
  2. Koziol, M. J., Bradshaw, C. R., Allen, G. E., Costa, A. S., Frezza, C. and Gurdon, J. B. (2016). Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nat Struct Mol Biol 23(1): 24-30.

简介

原代小胶质细胞,在单一培养或与神经元或星形胶质细胞共培养,是研究在中枢神经系统(CNS)中小胶质炎症反应和细胞类型特异性相互作用的机制的强大工具。这个协议提供了如何从新生小鼠幼崽制备高纯度原代小胶质细胞的细节。总的步骤包括脑细胞解离,混合胶质细胞培养和小胶质细胞分离。

[背景] 近年来,神经炎症已成为神经科学研究的热点领域。在患有各种神经疾病的患者的脑中观察到炎症反应,例如神经胶质激活和细胞因子上调(Fan等人,2015; Koshimori等人,2015;花园和坎贝尔,2016)。神经炎症不仅被认为是脑中病理变化的结果,而且也是疾病进展的原因(Schwartz等人,2013)。此外,炎症通路的生理功能,其重要性以前被低估,正被揭示为惊人的多才多艺。例如,补体信号通路的激活通常在神经疾病的中枢神经系统(CNS)中观察到,并且被怀疑参与疾病病理生理学(Michailidou等人,2015; Loeffler >等人,2008)。现在我们知道它也在突触精修的发育调节中起重要作用(Stevens等人,2007)。随着对炎症的越来越多的关注,对发展中的小胶质细胞功能,神经保护和发病机理的兴趣继续增长。小胶质细胞是位于脑中的骨髓谱系的驻留先天免疫细胞,并且是CNS中免疫系统的关键组分。在一些神经疾病中小胶质细胞的激活可以直接参与病原过程。例如,仅影响小胶质细胞的TREM2突变是阿尔茨海默氏病的遗传风险因子(Yuan等人,2016; Wang等人,2015)。同时,小胶质细胞的发育作用正在被揭示。例如,在早期发育期间的突触成熟需要小胶质细胞,并且这种调节可以强调发育疾病例如自闭症的发病机制(Edmonson等人,2016; Stephan等人 2012)。用于研究小胶质细胞的工具包括体内模型(例如,小胶质细胞缺陷型PU.1敲除小鼠[McKercher等人,1996])和体外系统如永生化小胶质细胞系和原代小胶质细胞培养物。虽然体内工具对于展示系统性小胶质细胞功能是强大的,但是体外工具由于容易操纵实验因素而是机械表征的理想选择。与永生化小胶质细胞系相比,原代小胶质细胞更好地模拟体内小胶质性质(Stansley等人,2012)。刺激后改变的基因表达可以在原代小胶质细胞中比在小胶质细胞系中更好地呈现(Stansley等人,2012; Henn等人,2009)。在这里我们描述了一种用于建立源自新生小鼠的高纯度原代小胶质细胞培养物的方案,并且该方法在我们的工作中产生了鲁棒的结果(Lian等人,2016)。通过收集的脑的酶消化收集解离的细胞并接种以生长混合胶质细胞培养物。通过机械敲击混合胶质细胞培养物来纯化在汇合星形胶质细胞层顶部生长的小胶质细胞。...

材料和试剂

  1. 微量离心管,1.7ml(Coring,Axygen ,目录号:MCT-175-C)
  2. 1.5ml带盖的Bioruptor Pico微管(Diagenode,目录号:C30010016)
  3. 可选:D1000胶带和试剂(Agilent Technologies,目录号:5067-5582; 5067-5583)
  4. DNeasy Blood& Tissue Kit(QIAGEN,目录号:69506)
  5. 磷酸盐缓冲盐水(PBS)
  6. CutSmart缓冲液(New England Biolabs,目录号:B7204S)
  7. RNase(无DNA酶)(Roche Diagnostics,目录号:11119915001)
  8. 水(已经高压灭菌的双去离子水)
    注意:本协议中使用的所有水都是指双重去离子水,随后进行高压灭菌。或者,可以使用购买的DNAse游离水。
  9. dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32854)
  10. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3059)
  11. dA 6m 抗体(Synaptic Systems,目录号:202011/202111/202003)
  12. Dynabeads 蛋白A(Thermo Fisher Scientific,Novex TM ,目录号:10008D)
  13. 糖原(Roche Diagnostics,目录号:10901393001)
  14. NaOAc(Thermo Fisher Scientific,Fisher Scientific,目录号:10010500)
  15. 异丙醇(Thermo Fisher Scientific,Fisher Scientific,目录号:10315720)
  16. 乙醇绝对(VWR,目录号:20821330)
  17. TruSeq Nano DNA LT文库制备试剂盒(A或B组)(Illumina,目录号:FC-121-4001或FC-121-4002)
  18. Tris-HCl(Melford Laboratories,目录号:B2005)
  19. 氯化钠(NaCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:10428420)
  20. Igepal CA-630(Sigma-Aldrich,目录号:I8896)
  21. N 6 - 甲基腺苷5'-单磷酸钠盐(Sigma-Aldrich,目录号:M2780)
  22. EDTA(Thermo Fisher Scientific,Fisher Scientific,目录号:10213570)
  23. 干冰
  24. 5x DIP IP缓冲区(参见配方)
  25. 1x DIP IP缓冲区(参见配方)
  26. DIP洗脱缓冲液(参见配方)

设备

  1. 孵化器(格兰特,干式加热系统)
  2. 旋转轮(Bibby Scientific,Stuart,型号:旋转器SB3)
  3. 热混合器(Eppendorf,型号:Thermomixer compact)
  4. Qubit荧光计(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32857)
    注意:此产品已停产。
  5. 水浴超声仪(Diagenode,Bioruptor Pico,目录号:B01060001)
  6. 微量离心机(Eppendorf,型号:Centrifuge 5424)
  7. Vortex(Scientific Industries,型号:Vortex-Genie 2)
  8. 磁铁(Thermo Fisher Scientific,DynaMag TM ,目录号:12321D)
  9. 可选:2200 Tapestation(安捷伦科技公司,目录号:G2965A)

程序

该程序可以分为三个部分:'A'分离基因组DNA,'B'超声和'C'DNA免疫沉淀dA <6m 。程序的概要如图1所示。我们鼓励使用至少两个生物复制品和至少2种不同的dA 6m抗体进行所有实验。作为进一步的对照,可以在E上进行DNA免疫沉淀过程。大肠杆菌细菌DNA,如原始工作(Koziol等人,2015)中所述。野生型细菌可以用作阳性对照,因为它们的dA 6m 位点是公知的。作为阴性对照,可以使用Dam dA 6m 甲基化酶缺陷的细菌。这样的细菌在序列GATC内不再具有脱氧腺苷甲基化,并且总体上具有减少数目的dA 6m位点。然后可以通过观察其dA 6m信号中的两个细菌基因组之间的显着差异,特别是通过dA 的减少来获得dA 6m 的DNA免疫沉淀的成功> <6m (Koziol等人,2015)。


图1. dA 6m DNA免疫沉淀的图示。分离基因组DNA,通过RNA酶处理消除任何RNA。接下来,使用针对dA 6m的抗体,通过DNA免疫沉淀将DNA片段化和富集。然后可以通过随后的深度测序分析富集的级分。

  1. 基因组DNA分离
    1. 使用DNeasy Blood&组织试剂盒,按照制造商的指导,但有例外:用177μlPBS,而不是AE缓冲液(10mM Tris-Cl,0.5mM EDTA,pH9.0)洗脱DNA。接下来,再次向柱中加入177μlPBS,并洗脱。收集两个部分在同一管,使总共354微升。
      注意:任何提取基因组DNA的方法都可以在该步骤中使用,只要获得至少5μg的DNA即可。如果使用另一种基因组DNA提取方法,请确保稀释基因组DNA,使最终体积为354μl。在该体积中,DNA的浓度应至少为14ng /μl。
    2. 加入40μlCutSmart缓冲液。
    3. 添加6微升的RNase DNase-free。
    4. 在37℃孵育过夜。
      注意:孵育至少6小时,以确保RNA的良好降解。 RNA含有甲基化的腺苷,抗体也可以识别。因此,RNA的存在可能抑制甲基化脱氧腺苷从DNA的回收,使得去除是重要的。
    5. 将样品分成两份,并清理基因组DNA:使用DNeasy Blood& Tissue Kit,按照制造商的方案"DNeasy Blood& Tissue Kit',用177μlPBS代替AE缓冲液洗脱DNA一次。
      注意:此柱清洁步骤有助于更好地从样品中消除RNA。需要拆分为两列,因为否则需要加载的卷超过列容量。
    6. 合并两个相应的样品。
    7. 加入40μlCutSmart缓冲液。
    8. 加入2μl无RNase DNA酶
    9. 在37℃孵育至少2小时 注意:此额外的RNase步骤可确保大部分RNA被降解。
    10. 将样品分成两份,并清理基因组DNA:使用DNeasy Blood& Tissue Kit,按照制造商的指南,用200μl水代替AE缓冲液洗脱DNA一次。
      注意:需要拆分为两列,否则需要加载的卷超过列容量。
    11. 合并两个相应的样品。
    12. 按照制造商的指南,使用Qubit荧光计和Qubit dsDNA HS测定试剂盒定量基因组DNA含量。
      注意:我们建议不要使用Nanodrop进行定量,因为这不会导致可靠的定量。在这一点上,可以停止方案并且可以冷冻DNA。我们建议在-20°C或更冷的储存。

  2. 基因组DNA的超声处理
    1. 取5微克的基因组DNA,并转移到1.5毫升Bioruptor Pico微管。
    2. 用水调节体积至300μl。
    3. 在4℃下,在Diagenode Bioruptor Pico中超声处理35个循环,30秒开/30秒关闭。一个代表性的例子如图2所示。
      注意:根据所使用的机器和基因组DNA,需要优化超声处理时间。我们的目标是使DNA片段大小达到峰值在约150-300个碱基对。
    4. 使用D1000胶带和试剂,按照制造商的方案验证基因组DNA与Tapestation的片段化 注意:作为Tapestation的替代,基因组DNA可以在琼脂糖凝胶上运行,以在超声处理后验证片段大小。

  3. dA 6m DNA免疫沉淀
    1. 取每个样品300 ng的超声处理的基因组DNA。这将是深度测序的输入控制,并且不会用于下述的DNA免疫沉淀过程。储存于-20°C直至需要
    2. 取3μg的超声处理的基因组DNA,并加入到1.7ml微量离心管中。
    3. 将管置于冰上。
    4. 在冰上,加入200μl5x DIP IP缓冲液
    5. 在冰上,加入200μl30μg/μlBSA储备液
    6. 在冰上,用水将总体积调节至1ml
    7. 短暂涡旋约2秒,然后放在冰上
    8. 加入5μl0.5μg/μldA 6m 抗体原液
    9. 立即通过转动管子并将管子放回冰上轻轻混合。
    10. 通过涡旋混合磁性蛋白A珠,使所有珠重新悬浮
    11. 取100微升混合磁性蛋白A珠每个样品,并添加到新的1.7毫升微量离心管
    12. 放在磁铁上,等待1分钟,磁珠粘在管子一侧。
    13. 从珠子上除去上清液。
    14. 从磁铁上去除珠子。
    15. 向珠子中加入1ml 1x DIP IP缓冲液,轻轻混匀
    16. 重复步骤C11-C14至少两次以上。
    17. 放在磁铁上,等待1分钟,磁珠粘在管子一侧。
    18. 从珠子上除去上清液。
    19. 将珠子重悬于105μl1x DIP IP缓冲液中
    20. 加入105μl30μg/μlBSA储备液。
    21. 在4℃下将珠子分别在转轮上孵育。
      注意:珠子和样品在单独的试管中孵育。
    22. 将样品分别在4℃的转轮上孵育。
    23. 过夜孵育后,向每个样品中加入200μl珠悬浮液
    24. 将样品珠混合物在4℃下在旋转轮上孵育至少一个多小时
    25. 将样品放在磁铁上,等待1分钟,磁珠粘在管子一侧。
    26. 清除珠粒上的所有上清液。
    27. 从磁铁上去除珠子。
    28. 向珠子中加入1ml 1x DIP IP缓冲液,轻轻混匀
    29. 重复步骤C23-C26至少三次以上。
    30. 将样品放在磁铁上,等待1分钟,磁珠粘在管子一侧
    31. 除去珠粒中的所有上清液。
    32. 加入200μlDIP洗脱缓冲液。
    33. 漩涡
    34. 将样品在42℃下在Thermomixer中至少1小时,以1400rpm
    35. 向下旋转样本。
    36. 将样品放在磁铁上,等待1分钟,磁珠粘在管子一侧
    37. 将上清液从珠子转移到新的1.7毫升微量离心管中 注意:您可以丢弃珠子。
    38. 向回收的上清液中加入300μl水
    39. 加入2微升糖原。
    40. 加入50μl的3M NaOAc的储备液。
    41. 加入500μl异丙醇。
    42. 漩涡
    43. 置于干冰上直至冷冻。
    44. 最高速度离心30分钟。
      注意:沉淀应该形成,其含有dA 6m 富集的DNA。
    45. 除去上清液。
    46. 用1ml 70%乙醇洗涤沉淀两次。
    47. 清除任何液体。
    48. 让颗粒在室温下干燥5分钟
    49. 重新悬浮在所需量的水中。
      注意:我们通常在约10-50微升
      重悬
    50. 按照制造商的指南,使用Qubit荧光计和Qubit dsDNA HS测定试剂盒定量基因组DNA含量。
      注意:此步骤是必需的,以确保您已实际获得基因组DNA与下拉。我们通常希望获得至少10 ng的基因组DNA。
    51. 使用步骤C47的所有基因组DNA和步骤C1的相应输入对照制备标准深度测序文库 注意:尽管任何标准深度测序都是可能的,但我们建议使用TruSeq Nano DNA LT文库制备试剂盒(A或B组)用于制备适合Illumina测序的文库,例如HiSeq 2500或HiSeq 4000。

数据分析

在已经应用该方法的原始研究论文中提供了关于数据处理和分析,重复,独立实验,可重复性(包括所应用的任何统计学测试)的任何信息(Koziol等人,2015) 。

代表数据




图2.超声处理后基因组DNA断裂模式的分布。使用Diagenode超声仪,将5μg基因组DNA在300μl的体积中片段化35分钟。通过Tapestation分析,用D1000 Tapestation试剂测定基因组DNA的大小。在该样品中,断裂的DNA的峰被确定为245bp。还示出了下部和上部标记。

笔记

  1. 我们已经发现这个协议是非常鲁棒和可重复的。我们总是能够富集dA 6m 并且为随后的标准深度测序文库获得总是足够的DNA。如果该方法不能产生足够的或没有DNA,我们没有遇到过,请重复实验,也许用新鲜的抗体批次。
  2. 我们测试了3种不同的dA 6m抗体用于在真核基因组中富集dA 6m 。所有这些都在上面列出。我们已经发现,它们都具有可重复和重叠的结果(Koziol等人,2015)。因此,我们相信任何这些抗体都会工作。然而,我们鼓励使用至少两种不同的dA 6m 抗体来确认任何初始结果,以对结果具有更高的置信度。

食谱

注意:在开始之前准备储备溶液中所有的新鲜溶液,并保持冰冻。

  1. 5x DIP IP缓冲区
    加入指定量至7.5ml水
    0.5ml 1M Tris-HCl(pH7.4)贮液 1.5ml 5M NaCl储液
    0.5ml 10%vol/vol Igepal CA-630原料
  2. 1x DIP IP缓冲区
    将10ml 5x DIP IP缓冲液与40ml水混合
  3. DIP洗脱缓冲液
    为每个样品添加指示量,根据样品数量
    按比例增加 45μl5×DIP IP缓冲液
    75μl20mM N 6 - 甲基腺苷5'-单磷酸钠盐储液
    105微升水

致谢

该方案用于以前在Nature Structural and Molecular Biology(Nature Science and Molecular Biology)(Koziol等人,2015)中发表的工作。 M.J.K.得到长期人类前沿奖学金(LT000149/2010-L),医学研究委员会奖学金(G1001690)和以撒牛顿信托奖学金(RG76588)的支持。该工作由生物技术和生物科学研究委员会授予BB/M022994/1(M.J.K.)赞助。进行这项工作的Gurdon实验室由来自Wellcome Trust的101050/Z/13/Z资助,由Gurdon Institute核心资助,即惠康信托核心资助(092096/Z/10/Z)和Cancer Research UK Grant(C6946/A14492)。 C.R.B.和G.E.A.由Wellcome Trust核心赠款资助。我们感谢S. Moss和K. Harnish对深度测序的建议和帮助,他们都得到了Gurdon Institute核心资助。我们感谢Nigel Garrett的批评性评论。本文报道的该方案是从用于检测RNA中腺苷甲基化的现有方法建立的(Dominissini等人,2013)。

参考文献

  1. Dominissini,D.,Moshkovitz,MS,Divon,SM,Amariglio,N.and Rechavil,G。(2013)。  - 甲基腺苷通过基于免疫捕获和大规模平行测序的 A-seq。 Nature Protocols 8(1):176-189。
  2. Koziol,MJ,Bradshaw,CR,Allen,GE,Costa,AS,Frezza,C.and Gurdon,JB(2016)。  在脊椎动物中甲基化脱氧腺苷的鉴定显示出DNA修饰的多样性。 Nat Struct Mol 23(1):24-30
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Koziol, M. J., Bradshaw, C. R., Allen, G. E., Costa, A. S. and Frezza, C. (2016). Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation. Bio-protocol 6(21): e1990. DOI: 10.21769/BioProtoc.1990.
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