搜索

Library Construction for Genome-wide Bisulfite Sequencing in Plants
拟南芥Bs-seq文库的构建   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.

Materials and Reagents

  1. DNA LoBind 1.5 and 2.0 ml centrifuge tubes (Eppendorf, catalog number: 22431021 and 22431048 , respectively)
  2. AMPure magnetic beads (Beckman Coulter, catalog number: A63881 )
  3. Ethanol (70% v/v, ethanol/water)
  4. TruSeq DNA sample preparation kit (Illumina, catalog number: FC-121-2001 )
  5. SYBR Safe (Life Technologies, catalog number: S33102 )
  6. 50 bp ladder (Thermo Fisher Scientific, catalog number: SM0373 )
  7. Disposable sterile scalpels (Thermo Fisher Scientific catalog number 12-460--452 )
  8. FalconTM 15 ml conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 14-959-49B )
  9. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
  10. Qubit dsDNA HS assay kit (Life Technologies, catalog number: Q32851 )
  11. EpiTect bisulfite kit (QIAGEN, catalog number: 59104 )
  12. MinElute PCR purification kit (QIAGEN, catalog number: 28004 )
  13. PfuTurbo Cx hotstart DNA polymerase (Agilent, catalog number: 600410-51 )
  14. KAPA library quantification kit (Kapa Biosystems, catalog number: KK4824 )
  15. TE buffer (see Recipes)
  16. Elution buffer (see Recipes)

Equipment

  1. S2 adaptive focus acoustic disruptor (Covaris)
  2. Microtubes with AFA fiber with pre-slit snap-caps (Covaris, catalog number: 520045 )
  3. Refrigerated water bath circulator (Thermo Fisher Scientific, model: SC100-A10 )
  4. Thermomixer R (Eppendorf, catalog number: 022670107 )
  5. MiniSpin PlusTM centrifuge (Eppendorf, catalog number 0 22620100 )
  6. Magnetic stand (Life Technologies, catalog number 4457858 )
  7. Vortex mixer (Thermo Fisher Scientific, catalog number 02-215-365 )
  8. 2100 BioAnalyzer (Agilent, catalog number G2943CA )
  9. Qubit 2.0 fluorometer (Life Technologies, catalog number Q32866 )
  10. Horizon 11-14, horizontal gel electrophoresis system (Apogee Designs, model: 11068020 )
  11. Safe Imager 2.0 blue light transilluminator (Life Technologies, model: G6600 )
  12. ABI7900HT real-time PCR system (Life Technologies)
  13. Thermal cycler (C1000 Touch) (Bio-Rad Laboratories, model: 185-1196EDU )
  14. Pipettors (RAININ, various models)

Procedure

The overall workflow of the procedure is illustrated in Figure 1.


Figure 1. Workflow of library construction for genome-wide bisulfite sequencing

  1. DNA fragmentation by ultrasonic destruction
    1. Dissolve ~2 μg of high quality, high molecular weight genomic DNA extracted from Arabidopsis leaf tissues in 130 μl TE buffer and transfer to a 6 x 16 mm glass microtube with AFA fiber with pre-slit snap-caps. Genomic DNA extracted using the commonly used CTAB (cetyltrimethyl ammonium bromide) method is suitable as long as the genomic DNA runs at a high molecular weight (> 12 kb) and as a clean single band in 0.8% agarose gel. The quality of genomic DNA can also be monitored with the 2100 BioAnalyzer (Figure 2A).


      Figure 2. BioAnalyzer traces of an Arabidopsis DNA sample (OE) that was processed through the workflow of the procedure described in this protocol. A. Genomic DNA; B. Ultrasonically fragmented DNA; C. Ultrasonically fragmented and magnetic bead cleaned up DNA; D. Adaptor-ligated DNA fragments recovered by the agarose gel-based selection; E. The final sequencing library (post PCR amplification).

    2. Shear the genomic DNA into fragments with an average size of 300 bp in the S2 adaptive focus acoustic disruptor using the following settings (for fragmenting DNA with the majority of fragments in the 150-450 bp range) (Figure 2B):
      Duty cycle 10%
      Intensity 4
      Cycles/burst 200
      Time 132 sec
      Temperature 6 °C
      The S2 instrument is connected to the refrigerated water bath circulator that is set at 6 °C in order to maintain a constant temperature during the sonication process.
    3. Transfer to a 1.5 ml microcentrifuge tube, and estimate the recovered volume with a pipettor. Typically, 125 μl of the DNA solution is recovered.
    4. Add a suspension (slur) of AMPure magnetic beads at a bead-to-sample ratio of 0.73:1 (v/v). This bead-to-sample ratio is used to collect DNA fragments over 200 bp prior to construction of the sequencing library.
    5. Incubate the mix at room temperature for 15 min with shaking at 700 rpm in the Thermomixer R to allow DNA to bind with the beads.
    6. Pulse spin to collect the mix at the bottom of the tube, and then put the tube on the magnetic stand for 1-2 min until the solution is completely clear. The magnetic beads will be attracted to the magnet, thus allowing removal of the supernatant. Remove and discard the supernatant.
    7. Leave the tube on the magnetic stand and wash the beads twice as follows: for each wash add 1 ml of freshly prepared 70% ethanol, gently pipet the beads up and down six times, let the beads sit for 30-60 sec, and then discard the supernatant by pipetting without disturbing the beads.
    8. Following the second wash, pulse spin the tube to collect traces of ethanol, and then place the tube on the magnetic stand. Remove ethanol traces with a fine-tipped pipettor.
    9. Air dry the magnetic beads for 1 to 3 min at room temperature.
    10. Elute DNA as follows: remove the tube from the magnetic stand and place on a plastic rack, add 30 μl of elution buffer, vortex vigorously for 10 sec, place the tube back on the magnetic stand until the solution becomes completely clear, and then use a pipettor to transfer the supernatant containing the DNA into a clean microcentrifuge tube. Repeat the elution by adding another 30 μl of elution buffer as above. Pool the two eluate fractions.
    11. Save 3 μl of the DNA sample for quantification in the Qubit 2.0 fluorometer according to the product instructions (http://www.lifetechnologies.com/order/catalog/product/Q32854) and for fragment size confirmation with the 2100 BioAnalyzer (Figure 2C). The desired size range of the fragments should be 200-450 bp.
      At this point the DNA can be stored at -20 °C for later use.

  2. Library construction
    Sequencing libraries are made using the low-throughput TruSeq DNA sample preparation kit following the manufacturer's protocol (TruSeq DNA sample preparation guide) with a few modifications (see below). The adaptors included in this kit are methylated and are therefore compatible with bisulfite sequencing for DNA methylation analysis.
    1. End repair the DNA fragments in the following reaction:
      50 μl above DNA sample (~1.5 μg)
      10 μl end-repair reaction buffer (included in the TruSeq Kit)
      40 μl end-repair mix (included in the TruSeq Kit)
      Incubate at 30 °C for 30 min. Clean up the reaction mix with AMPure magnetic beads as described above (DNA Fragmentation section: steps A4-10), except that the bead-to-sample ratio is 1:1 (v/v). Elute DNA in 17.5 μl elution buffer. This bead-to-sample ratio is used to collect essentially all DNA fragments.
    2. 3’-adenylate the end-repaired DNA fragments in the following reaction:
      15 μl end-repaired DNA
      2.5 μl 3'-adenylation reaction buffer (included in the TruSeq Kit)
      12.5 μl A-tailing mix (included in the TruSeq Kit)
      Incubate at 37 °C for 30 min. The adenylation mix is compatible with the ligation reaction; therefore, no clean-up is necessary at this step.
    3. Ligate methylated adaptors to the 3'-adenylated DNA fragments in the following reaction:
      30 μl adenylated DNA (the entire adenylation reaction from step B2)
      2.5 μl ligation reaction buffer (included in the TruSeq Kit)
      2.5 μl ligation mix (included in the TruSeq Kit)
      2.5 μl adaptors (included in the TruSeq Kit)
      Incubate at 30 °C for 10 min. Add 5 μl of ligation stop buffer. Clean-up sample using AMPure magnetic beads as described above (DNA Fragmentation section: steps A4-10). with a 1:1 (v/v) bead-to-sample ratio. Elute adaptor-ligated DNA in 22.5 μl elution buffer.
    4. Size-select adaptor-ligated DNA fragments (250-500 bp) in a 2% TAE agarose gel as follows. Prepare 100 ml of the solution in a 500 ml Erlenmeyer flask, heat in a microwave oven until agarose is fully dissolved, add SYBR Safe stain (1:10,000 v/v dye-to-gel ratio) to the molten agarose solution and swirl to mix, and then pour the gel-dye solution into properly set-up casting of the gel electrophoresis apparatus. A 10-well comb is placed at one end of the gel casting tray to form wells. Allow gel to fully solidify (~30 min). Load a 50 bp ladder in one of the gel wells to aid in size estimation. Mix the sample with a suitable sample loading dye and load into any of the additional wells. Connect electrodes to power supply and electrophorese at 100 V until the quickest moving dye reaches 3/4 of the gel length from the wells. No post electrophoresis staining is necessary when the dye is added as suggested here. SYBR Safe is used instead of ethidium bromide in order to avoid UV damage (during viewing) of DNA fragments. View the gel on a Safe Imager 2.0 blue light transilluminator. Cut the gel region containing fragments in the 250-500 bp range using a sterile, disposable scalpel (Figure 3). Transfer the gel slice into a DNA LoBind 2.0 ml centrifuge tube. If gel slices are too large, they can be placed in a 15 ml conical tube. After the agarose gel is dissolved (see step 5 below), split dissolved volume if necessary into DNA LoBind 2.0 ml tubes for DNA extraction.


      Figure 3. Size selection of adaptor-ligated DNA fragments in an agarose gel. Two Arabidopsis genomic DNA samples (A and B) were fragmented in the S2 adaptive focus acoustic disruptor, end-repaired, 3’-adenylated, adaptor-ligated, and separated in a 2% TAE agarose gel. Gel wells were formed using a 10-well-forming comb (8 mm wide wells). Ten μl of ready-to-load 50 bp ladder were loaded in the flanking positions of the samples in the gel for excision process. Left: before excision; Right: post excision.

    5. Extract DNA from the gel using the QIAquick gel extraction kit following the manufacturer's instructions (http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/qiaquick-gel-extraction-kit#resources). Elute DNA with 22 μl elution buffer (included in the QIAquick gel extraction kit).
    6. Set aside 2 μl to quantitate the library in the Qubit 2.0 fluorometer with a Qubit dsDNA HS assay kit according to the product instructions (http://www.lifetechnologies.com/order/catalog/product/Q32854). The final library yield using this protocol is usually 300-400 ng (Figure 2D), which corresponds to a concentration of 13-18 DNA ng/ μl.
      At this point the DNA library can be stored at -20 °C for later use.

  3. Bisulfite treatment
    The entire library (300-400 ng) is subjected to sodium bisulfite treatment using the EpiTect bisulfite kit following the manufacturer's instructions (http://www.qiagen.com/products/catalog/assay-technologies/epigenetics/epitect-bisulfite-kits - resources). Briefly:
    1. Prepare buffer BW, buffer BD, carrier RNA, buffer BL, and bisulfite mix following the instructions in the kit.
    2. Treat DNA with bisulfite in the following reaction:
      20 μl DNA (the entire library)
      85 μl bisulfite mix (included in the EpiTect kit)
      35 μl DNA protect buffer (included in the EpiTect kit)
      Incubate at the following conditions in a thermal cycler: 95 °C for 5 min, 60 °C for 25 min, 95 °C for 5 min, 60 °C for 85 min, 95 °C for 5 min, 60 °C for 175 min, and finally hold at 20 °C.
    3. Purify the bisulfite-converted DNA following the manufacturer's instructions. Elute DNA twice, each time with 20 μl elution buffer (included in the EpiTect kit) and pool the two eluate fractions.
    4. Further purify and concentrate the DNA using a MinElute column following the instructions in the MinElute PCR purification kit (http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/minelute-pcr-purification-kit#resources). Elute DNA in 15 μl elution buffer (included in the MinElute PCR purification kit).
      At this point the bisulfite-converted library can be stored at -20 °C for later use.

  4. Library enrichment
    1. Amplify ~1 ng of the bisulfite-converted library using a uracil-insensitive PfuTurbo Cx hotstart DNA polymerase and the primer cocktail included in the Illumina TruSeq library construction kit in the following reaction:
      17.25 μl double-distilled water
      15 μl DNA template (~1 ng, usually the entire eluate of the above library)
      5 μl Pfu Turbo Buffer (coming with the Pfu Turbo polymerase)
      2.5 μl DMSO
      1.25 μl dNTPs (10 mM)
      5 μl primer cocktail (included in the TruSeq Kit)
      1μl Pfu Turbo Cx Hotstart polymerase
      The amplification conditions are: denaturation at 98 °C for 30 sec, 14-18 cycles (98 °C for 15 sec, 60 °C for 30 sec, and 72 °C for 1 min), and final extension at 72 °C for 5 min. Depending on the yield of the bisulfite treatment step, the number of PCR amplification cycles should be as small as possible. This step is to selectively enrich DNA library fragments that have methylated adaptors at both ends, and to generate sufficient DNA for accurate quantitation.
    2. Clean the PCR amplification products twice using AMPure magnetic beads as describe above (DNA Fragmentation section: steps A4-10) with a bead-to-sample ratio of 0.85:1 (v/v). Elute DNA in 30 μl of elution buffer. This bead-to-sample ratio is used to remove dNTPs and primers while retaining all the DNA amplification fragments above 150 bp.
    3. Use 1 μl of the DNA sample for fragment size confirmation with the 2100 BioAnalyzer. The average insert size of the library generated by this protocol is ~360 bp (Figure 2E). A different average size can be obtained by adjusting the DNA ultrasonic fragmentation conditions (DNA fragmentation section: step A2) and by choosing the desired range at the size selection step (Library construction section: step B4).
    4. Use 1 μl of the DNA sample for quantification in the Qubit 2.0 fluorometer and 1 μl for quantification by qPCR with the KAPA library quantification kit on the ABI7900HT real-time PCR system. This amplification protocol results in 50-200 ng of library in ~26 μl.
      The library is now ready for sequencing. If necessary, the library concentration is adjusted to 10 nM by diluting with Elution buffer (see recipes) and submitted for sequencing.

Recipes

  1. Elution buffer
    10 mM Tris, pH 8.0
  2. TE buffer
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA

Acknowledgments

The protocol was adapted from Wang et al. (2013). This work was supported by a grant from the National Science Foundation (IOS-0842716) awarded to Z.M.

References

  1. Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H. and Ecker, J. R. (2008). Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133(3): 523-536.
  2. TruSeq DNA sample preparation guide (PE-940-2001)
    (http://nextgen.mgh.harvard.edu/attachments/TruSeq_DNA_SamplePrep_Guide_15005180_A.pdf
  3. Wang, Y., An, C., Zhang, X., Yao, J., Zhang, Y., Sun, Y., Yu, F., Amador, D. M. and Mou, Z. (2013). The Arabidopsis Elongator complex subunit2 epigenetically regulates plant immune responses. Plant Cell 25(2): 762-776. 

简介

DNA甲基化是研究最多的表观遗传修饰,其涉及将甲基添加到DNA中胞嘧啶残基的碳-5位置。 DNA甲基化对于基因表达的调节是重要的。 亚硫酸氢盐测序是确定真核生物全基因组DNA甲基化谱的金标准技术。 该协议描述了如何在拟南芥中制备用于全基因组亚硫酸氢盐测序的基因组DNA文库,其可以适用于其他植物物种。

材料和试剂

  1. DNA LoBind 1.5和2.0ml离心管(Eppendorf,目录号分别为22431021和22431048)
  2. AMPure磁珠(Beckman Coulter,目录号:A63881)
  3. 乙醇(70%v/v,乙醇/水)
  4. TruSeq DNA样品制备试剂盒(Illumina,目录号:FC-121-2001)
  5. SYBR Safe(Life Technologies,目录号:S33102)
  6. 50b阶梯(Thermo Fisher Scientific,目录号:SM0373)
  7. 一次性无菌手术刀(Thermo Fisher Scientific目录号12-460-452)
  8. Falcon TM 15ml锥形离心管(Thermo Fisher Scientific,目录号:14-959-49B)
  9. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  10. Qubit dsDNA HS测定试剂盒(Life Technologies,目录号:Q32851)
  11. EpiTect亚硫酸氢盐试剂盒(QIAGEN,目录号:59104)
  12. MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
  13. PfuTurbo Cx hotstart DNA聚合酶(Agilent,目录号:600410-51)
  14. KAPA文库定量试剂盒(Kapa Biosystems,目录号:KK4824)
  15. TE缓冲区(参见配方)
  16. 洗脱缓冲液(见配方)

设备

  1. S2自适应聚焦声波破碎器(Covaris)
  2. 具有AFA纤维的微管与预切割卡扣帽(Covaris,目录号:520045)
  3. 冷冻水浴循环器(Thermo Fisher Scientific,型号:SC100-A10)
  4. Thermomixer R(Eppendorf,目录号:022670107)
  5. MiniSpin Plus TM sup/TM离心机(Eppendorf,目录号022620100)
  6. 磁性架(Life Technologies,目录号4457858)
  7. 涡旋混合器(Thermo Fisher Scientific,目录号02-215-365)
  8. 2100 BioAnalyzer(Agilent,目录号G2943CA)
  9. Qubit 2.0荧光计(Life Technologies,目录号Q32866)
  10. Horizon 11-14,水平凝胶电泳系统(Apogee Designs,型号:11068020)
  11. Safe Imager 2.0蓝光透射仪(Life Technologies,型号:G6600)
  12. ABI7900HT实时PCR系统(Life Technologies)
  13. 热循环仪(C1000 Touch)(Bio-Rad Laboratories,型号:185-1196EDU)
  14. 移液器(RAININ,各种型号)

程序

该过程的整体工作流程如图1所示。


图1.全基因组亚硫酸氢盐测序的文库构建工作流程

  1. 通过超声破坏DNA片段
    1. 将从拟南芥叶组织中提取的〜2μg高质量,高分子量基因组DNA溶解在130μlTE缓冲液中,并转移至具有AFA纤维的6×16mm玻璃微管中,具有预狭缝卡口。使用常用的CTAB(十六烷基三甲基溴化铵)方法提取的基因组DNA是合适的,只要基因组DNA以高分子量(> 12kb)运行并且作为在0.8%琼脂糖凝胶中的纯单带。基因组DNA的质量也可以用2100 BioAnalyzer监测(图2A)

      图2.通过本协议中所述程序的工作流程处理的拟南芥DNA样品(OE)的BioAnalyzer痕迹。 A.基因组DNA; B.超声断裂的DNA;超声破碎和磁珠清除DNA; D.通过基于琼脂糖凝胶的选择回收的接头连接的DNA片段; E.最终测序文库(PCR后扩增)。

    2. 使用以下设置(用于片段化大多数片段在150-450bp范围内的DNA)将基因组DNA剪切成平均大小为300bp的片段在S2自适应焦点声学破碎器中(图2B):
      占空比10%
      强度4
      周期/突发200
      时间132秒
      温度6°C
      S2仪器连接到设定在6℃的冷却水浴循环器,以便在超声处理过程中保持恒定的温度。
    3. 转移到1.5毫升微量离心管,并用吸移管管理员估计回收的体积。通常,回收125μl的DNA溶液
    4. 以0.73:1(v/v)的珠子与样品比例加入AMPure磁珠的悬浮液(浆液)。在构建测序文库之前,这种珠子与样品之比用于收集超过200bp的DNA片段
    5. 在室温下孵育混合物15分钟,在Thermomixer R中以700rpm摇动,使DNA与珠结合。
    6. 脉冲旋转收集管底部的混合物,然后将管在磁力架上1-2分钟,直到溶液完全透明。磁珠将被吸引到磁体,从而允许去除上清液。取出并弃去上清液。
    7. 离开管在磁力架上,洗涤珠两次,如下:每次洗涤加入1毫升新鲜制备的70%乙醇,轻轻吸取珠上下六次,让珠子坐30-60秒,然后通过移液除去上清液而不干扰珠粒
    8. 在第二次洗涤后,脉冲旋转管以收集痕量的乙醇,然后将管置于磁性支架上。用细尖移液器移除乙醇痕迹。
    9. 在室温下将磁珠空气干燥1至3分钟。
    10. 洗脱DNA如下:从磁力架上取下试管,放在塑料架上,加入30μl洗脱缓冲液,剧烈涡旋10秒,将试管放回磁力架,直到溶液变得完全澄清,然后使用移液器将含有DNA的上清液转移到干净的微量离心管中。通过加入另外30μl如上的洗脱缓冲液重复洗脱。合并两个洗脱级分。
    11. 根据产品说明,将3μlDNA样品保存在Qubit 2.0荧光计中进行定量( http ://www.lifetechnologies.com/order/catalog/product/Q32854 )和用2100BioAnalyzer确认片段大小(图2C)。片段的所需大小范围应为200-450bp 此时,DNA可以储存在-20℃下备用
  2. 图书馆建设
    根据制造商的方案,使用低通量TruSeq DNA样品制备试剂盒制备测序文库( TruSeq DNA样品制备指南),并做了一些修改(见下文)。该试剂盒中包含的衔接头是甲基化的,因此与亚硫酸氢盐测序兼容,用于DNA甲基化分析
    1. 在以下反应中修复DNA片段:
      50μl以上DNA样品(〜1.5μg)
      10μl末端修复反应缓冲液(包含在TruSeq Kit中)
      40μl末端修复混合物(包含在TruSeq Kit中)
      在30℃孵育30分钟。除了珠与样品的比率为1:1(v/v)之外,如上所述用AMPURE磁珠清洁反应混合物(DNA断裂部分:步骤A4-10)。洗脱在17.5微升洗脱缓冲液中的DNA。这种珠子与样品比率用于收集基本上所有的DNA片段
    2. 在以下反应中将末端修复的DNA片段3'-腺苷酸化:
      15μl末端修复的DNA
      2.5μl3'-腺苷酸化反应缓冲液(包含在TruSeq试剂盒中) 12.5μlA-tail混合物(包含在TruSeq Kit中)
      在37℃孵育30分钟。腺苷酸化混合物与连接反应相容;因此,在这一步骤不需要清理
    3. 在以下反应中将甲基化的衔接子连接到3'-腺苷酸化的DNA片段:
      30μl腺苷酸化DNA(来自步骤B2的整个腺苷酸化反应) 2.5μl连接反应缓冲液(包含在TruSeq试剂盒中) 2.5μl连接混合物(包含在TruSeq试剂盒中) 2.5μl适配器(包含在TruSeq Kit中)
      在30℃孵育10分钟。加入5微升连接终止缓冲液。使用如上所述的AMPure磁珠清洁样品(DNA断裂部分:步骤A4-10)。具有1:1(v/v)珠子与样品比率。在22.5μl洗脱缓冲液中洗脱接头连接的DNA
    4. 如下在2%TAE琼脂糖凝胶中大小选择接头连接的DNA片段(250-500bp)。在500ml锥形烧瓶中制备100ml溶液,在微波炉中加热直至琼脂糖完全溶解,向熔融的琼脂糖溶液中加入SYBR安全染色(1:10,000v/v染料与凝胶比),并旋转混合,然后将凝胶 - 染料溶液倒入适当设置的凝胶电泳装置中。将10孔梳子放置在凝胶浇铸托盘的一端以形成孔。允许凝胶完全固化(〜30分钟)。在凝胶孔之一中加载50 bp梯度以帮助估计大小。将样品与合适的样品加载染料混合,并加载到任何额外的孔中。将电极连接到电源,并在100 V电泳,直到最快的移动染料达到3/4的凝胶长度从孔。当如本文所述加入染料时,不需要后电泳染色。使用SYBR Safe代替溴化乙锭以避免DNA片段的UV损伤(在观察期间)。在Safe Imager 2.0蓝光透射仪上观察凝胶。切割含有片段的凝胶区域 250-500 bp范围内使用无菌,一次性解剖刀(图3)。将凝胶切片转移到DNA LoBind 2.0ml离心管中。如果凝胶切片太大,它们可以放置在15ml锥形管中。琼脂糖凝胶溶解后(见下面的步骤5),如有必要将溶解体积分成DNA LoBind 2.0ml管用于DNA提取。


      图2.在琼脂糖凝胶中接头连接的DNA片段的大小选择将两个拟南芥基因组DNA样品(A和B)在S2自适应焦点声学破碎器中片段化,末端修复,3'-腺苷酸化,接头连接,并在2%TAE琼脂糖凝胶中分离。使用10孔形成梳(8mm宽孔)形成凝胶孔。将10μl即用载荷的50bp梯度装载在凝胶样品的侧翼位置用于切除过程。左:切除前;右:切除后。

    5. 使用QIAquick凝胶提取试剂盒,根据制造商的说明从凝胶中提取DNA( http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/qiaquick-gel-extraction-kit #resources )。用22μl洗脱缓冲液(包括在QIAquick凝胶提取试剂盒中)洗脱DNA
    6. 取出2μl,根据产品说明书用Qubit dsDNA HS测定试剂盒定量Qubit 2.0荧光计中的文库( http://www.lifetechnologies.com/order/catalog/product/Q32854 )。使用该方案的最终文库产量通常为300-400ng(图2D),其对应于13-18DNA ng /μl的浓度。
      此时,DNA文库可以储存在-20℃下备用
  3. 亚硫酸氢盐处理
    根据制造商的说明书( http://www.qiagen.com/products/catalog/assay-technologies/epigenetics/epitect-bisulfite-kits - resources )。简要地说:
    1. 按照试剂盒中的说明制备缓冲液BW,缓冲液BD,载体RNA,缓冲液BL和亚硫酸氢盐混合物
    2. 在以下反应中用亚硫酸氢盐处理DNA:
      20μlDNA(整个文库)
      85μl亚硫酸氢盐混合物(包含在EpiTect套件中)
      35μlDNA保护缓冲液(包含在EpiTect试剂盒中)
      在以下条件下在热循环仪中孵育:95℃5分钟,60℃25分钟,95℃5分钟,60℃85分钟,95℃5分钟,60℃175分钟min,最后保持在20℃
    3. 按照制造商的说明纯化亚硫酸氢盐转化的DNA。洗脱DNA两次,每次用20μl洗脱缓冲液(包括在EpiTect试剂盒中),并汇集两个洗脱液部分。
    4. 使用MinElute柱按照MinElute PCR纯化试剂盒中的说明进一步纯化和浓缩DNA( http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/minelute-pcr -purification-kit#resources )。在15μl洗脱缓冲液(包括在MinElute PCR纯化试剂盒中)洗脱DNA 此时,亚硫酸氢盐转化的文库可以储存在-20℃下备用
  4. 图书馆丰富
    1. 使用尿嘧啶不敏感的PfuTurbo Cx hotstart DNA聚合酶和Illumina TruSeq文库构建试剂盒中包含的引物混合物扩增约1ng亚硫酸氢盐转化的文库,反应如下:
      17.25μl双蒸水
      15μlDNA模板(〜1ng,通常是上述文库的整个洗脱液) 5μlPfu Turbo Buffer(含Pfu Turbo聚合酶)
      2.5μlDMSO
      1.25μldNTP(10mM) 5μl引物混合液(包含在TruSeq Kit中)
      1μlPfu Turbo Cx Hotstart聚合酶
      扩增条件为:98℃变性30秒,14-18个循环(98℃15秒,60℃30秒,72℃1分钟),最后在72℃延伸1次5分钟。根据亚硫酸氢盐处理步骤的产量,PCR扩增循环的数目应尽可能小。该步骤是选择性富集在两端具有甲基化衔接子的DNA文库片段,并产生用于精确定量的足够的DNA。
    2. 使用如上所述的AMPURE磁珠(DNA断裂部分:步骤A4-10)以0.85:1(v/v)的珠与样品比例清洗PCR扩增产物两次。洗脱DNA在30μl洗脱缓冲液中。这种珠子与样品之比用于去除dNTP和引物,同时保留所有DNA扩增片段超过150bp。
    3. 使用1微升的DNA样品用于2100 BioAnalyzer的片段大小确认。通过该方案产生的文库的平均插入大小为〜360bp(图2E)。通过调整DNA超声裂解条件(DNA断裂部分:步骤A2),并通过在大小选择步骤(文库构建部分:步骤B4)选择所需的范围,可以获得不同的平均大小。
    4. 使用1μlDNA样品在Qubit 2.0荧光计中定量,1μl用于通过qPCR用ABAP7900HT实时PCR系统上的KAPA文库定量试剂盒定量。 这种扩增方案产生50-200ng的库在〜26μl。
      该库现在已准备好进行测序。 如果需要,通过用洗脱缓冲液稀释(参见配方)将文库浓度调节至10nM,并提交测序。

食谱

  1. 洗脱缓冲液
    10mM Tris,pH8.0
  2. TE缓冲区
    10mM Tris-HCl,pH8.0 1mM EDTA

致谢

该方案改编自Wang等人(2013)。这项工作得到国家科学基金会(IOS-0842716)授予Z.M.的资助。

参考文献

  1. Lister,R.,O'Malley,R.C.,Tonti-Filippini,J.,Gregory,B.D.,Berry,C.C.,Millar,A.H。和Ecker,J.R。(2008)。 拟南芥中表观基因组的高度整合的单碱基分辨率图。/a> 电池 133(3):523-536。
  2. TruSeq DNA样品制备指南(PE-940-2001)
    http://nextgen.mgh.harvard.edu/attachments/TruSeq_DNA_SamplePrep_Guide_15005180_A.pdf) 
  3. Wang,Y.,An,C.,Zhang,X.,Yao,J.,Zhang,Y.,Sun,Y.,Yu,F.,Amador,D.M.and Mou,Z.(2013)。 拟南芥 Elongator复合亚基2表观遗传调节植物免疫反应。 植物细胞 25(2):762-776。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Amador, D. M., Wang, C., Holland, K. H. and Mou, Z. (2013). Library Construction for Genome-wide Bisulfite Sequencing in Plants. Bio-protocol 3(24): e1013. DOI: 10.21769/BioProtoc.1013.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。