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microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this protocol we successfully generated three dRNA-seq libraries using three solanaceae plants, including tobacco, tomato and potato. Although this protocol was developed with single-plexed adapter, it should be able to generate multiplexed libraries by replacing the 3’ adapter with multiplexing compatible 3’ adapter and replacing the PCR primer with indexed primers.

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Preparation of cDNA Library for dRNA-seq
用于dRNA-测序的cDNA文库的构建

植物科学 > 植物分子生物学 > RNA > RNA 测序
作者: Feng Li
Feng LiAffiliation 1: Department of Plant and Microbial Biology, Plant Gene Expression Center, University of California, Berkeley, USA
Affiliation 2: Agricultural Research Services (USDA-ARS), United States Department of Agriculture, Albany, USA
For correspondence: chdlifeng@mail.hzau.edu.cn
Bio-protocol author page: a180
 and Barbara Baker
Barbara BakerAffiliation 1: Department of Plant and Microbial Biology, Plant Gene Expression Center, University of California, Berkeley, USA
Affiliation 2: Agricultural Research Services (USDA-ARS), United States Department of Agriculture, Albany, USA
For correspondence: bbaker@berkeley.edu
Bio-protocol author page: a181
Vol 2, Iss 23, 12/5/2012, 5341 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.302

[Abstract] microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this protocol we successfully generated three dRNA-seq libraries using three solanaceae plants, including tobacco, tomato and potato. Although this protocol was developed with single-plexed adapter, it should be able to generate multiplexed libraries by replacing the 3’ adapter with multiplexing compatible 3’ adapter and replacing the PCR primer with indexed primers.
Keywords: NGS(NGS), Degradome(降解物组), MiRNA(miRNA), SiRNA(siRNA), Target(目标)

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. RNeasy Plant Mini Kit (QIAGEN, catalog number: 74903 )
  2. OligodT Dynabeads (Life Technologies, Invitrogen™, catalog number: 610-02 )
  3. SeaKem LE Agrose (Lonza, catalog number: 50004 )
  4. Illumina sRNA-seq 3’ adapter (Illumina, catalog number: 1000596 )
  5. RNase free water (Life Technologies, Invitrogen™, catalog number: 10977-023 )
  6. RNeasy Micro Kit (QIAGEN, catalog number: 74004 )
  7. Antarctic phosphatase (New England Biolabs, catalog number: M0289S )
  8. RNase OUT(Life Technologies, Invitrogen™, catalog number: 10777-019 )
  9. T4 RNA Ligase 1 (New England Biolabs, catalog number: M0204S )
  10. Illumina sRNA-seq RT primer (Illumina, catalog number: 1000597 )
  11. Illumina sRNA-seq 5’ adapter (Illumina , catalog number: 1000595 )
  12. Illumina sRNA-seq PCR primer (Illumina, catalog number: 1000591 , 1000592 )
  13. Gel purification kit (QIAGEN, catalog number: 28704 )
  14. dNTP (New England Biolabs, catalog number: N0447S )
  15. SuperScript II RT(Life Technologies, Invitrogen™, catalog number: 18064-022 )
  16. Zero Blunt® PCR Cloning Kit (Life Technologies, Invitrogen™, catalog number: K2700-40 )
  17. Agrose gel (Lonza)

Equipment

  1. PCR Thermal Cycler
  2. Illumina GA II sequencing system
  3. Pipette (20 μl, 200 μl, 1,000 μl)
  4. Magnetic bar

Procedure

  1. Isolation of high molecular weight RNA (with length > 200 bp) from plant tissue using RNeasy Plant Mini Kit according to manufacturer’s protocol (according to the manufacturer’s protocol, about 60 μg high molecular weight RNA can be obtained from 100 mg tobacco leaf tissue).
  2. Purification of polyA RNA from 10 μg of total RNA using OligodT Dynabeads according to manufacturer’s protocol and elute the polyA RNA in 15 μl RNase free water (a thermal cycler and a magnetic bar are used in this step).
  3. Ligate sRNA 5’ adapter:
    Purified mRNA
    14 μl
    sRNA 5’ adapter (10 μM)
    2 μl (Illumina sRNA-seq 5’ adapter)
    10x T4 RNA Ligase buffer
    2 μl (* If ATP is not included, add ATP to 1 mM final)
    T4 RNA Ligase I (10 U/μl)
    1.5 μl
    RNase OUT (40 U/μl) 
    0.5 μl
    20 °C, 6 h.
  4. Dynabeads purification and elute in 15 μl RNase free water according to manufacturer’s protocol.
  5. RNA fragmentation
    Fragmentation buffer
    1.6 μl (100 mM ZnCl2, 100 Mm Tris-HCl, pH7.0)
    Ligated mRNA
    14.4 μl
    70 °C 2.5 min
    Purify fragmented RNA using RNeasy Micro Kit and elute RNA in 17 μl RNase free water after purification.
  6. Phosphotase treatment to remove 3’ phosphate resulted from fragmentation:
    Fragmented RNA
    16 μl
    10x phosphatase buffer
    2 μl
    Antarctic phosphatase
    1 μl
    RNase OUT (40 U/μl)
    1 μl
    37 °C, 30 min
    4 °C hold
    Purify RNA by RNeasy Micro Kit and elute in 15 μl RNase free water.
  7. Ligate sRNA 3’ adaptor
    Purified RNA from step 6
    14.5 μl
    10x RNA Ligase buffer
    2 μl (* if ATP is not included, add ATP to 1 mM final)
    RNA Ligase 1 (10 Uμl)
    2 μl
    RNase OUT (40 U/μl)
    1 μl
    RNA adapter 3’ 0.5 μl
    (10 μM, Illumina sRNA-seq 3’ adapter)
    20 °C, 4 h
  8. Reverse transcriptation
    Prepare the following mix, heat at 70 °C for 2 min and place on ice.
    Adapter ligated RNA
    4 μl
    SRA RT primer
    0.5 μl (Illumina sRNA-seq RT primer)
    50 mM dNTP
    0.5 μl
    Prepare the following mix and add to the above reaction:
    5x first strand buffer
    2 μl
    100 mM DTT
    2 μl
    RNase OUT (40 U/μl)
    0.25 μl
    48 °C for 3 min then add:
    SuperScript II RT 0.75 μl
    44 °C for 60 min
  9. PCR amplification
    Prepare the following mix and add to RT reaction:
    Phusion HF 2x mix
    25 μl
    Primer GX1
    1 μl (Illumina sRNA-seq PCR primer)
    Primer GX2 
    1 μl (Illumina sRNA-seq PCR primer)
    Nuclease-free water
    13 μl
    Run the following protocol:
    1. 98 °C 30 sec
    2. 30-35 cycles of:
      98 °C 10 sec
      60 °C 30 sec
      72 °C 15 sec
    3. 72 °C 10 min
    4. 4 °C hold
  10. Run the PCR product through a 1.5% Agrose gel, cut a smear region between 150 bp and 250 bp and purify by Gel purification kit and elute in 25 μl elution buffer.
  11. Check the library quality by bio-analyzer High sensitivity DNA assay to check the size distribution (one μl sample is used in this step and a smear region centered around 250 bp is expected from the bio-analyzer electrophoresis profile, see Figure 1).




    Figure 1. Bioanalyzer analysis of dRNA-seq library. The upper part is the electrophoresis graph from the bio-analyzer run and the peak region between the two blue lines represents the purified dRNA constructs. Table below the electrophoresis graph shows the analysis of the peak region by the bio-analyzer 2100 software.

  12. Use Zero Blunt® PCR Cloning Kit to clone the library and sequence of a few clones to verify the presence of inserts derived from plant transcripts.
  13. Sequence the library using small RNA sequencing run on an Illumina GA II sequencing system.

Acknowledgments

The principle and application of this protocol were briefly described in Li et al. (2012). This work was supported by the National Science Foundation Plant Genome Research Program Grant (DBI-0218166) and the United States Department of Agriculture (CRIS 5335-22000-007-00D). The authors declare no conflict of interest.

References

  1. Li, F., Orban, R. and Baker, B. (2012). SoMART: a web server for plant miRNA, tasiRNA and target gene analysis. Plant J 70(5): 891-901.
    (Please cite this paper when you use this method in your publications)

材料和试剂

  1. RNeasy Plant Mini Kit(QIAGEN,目录号:74903)
  2. OligodT Dynabeads(Life Technologies,Invitrogen TM,目录号:610-02)

  3. 。SeaKem LE Agrose(Lonza,目录号:50004)
  4. Illumina sRNA-seq 3'衔接头(Illumina,目录号:1000596)
  5. 无RNA酶水(Life Technologies,Invitrogen TM,目录号:10977-023)
  6. RNeasy Micro Kit(QIAGEN,目录号:74004)
  7. 南极磷酸酶(New England Biolabs,目录号:M0289S)
  8. RNase OUT(Life Technologies,Invitrogen TM,目录号:10777-019)
  9. T4 RNA连接酶1(New England Biolabs,目录号:M0204S)
  10. Illumina sRNA-seq RT引物(Illumina,目录号:1000597)
  11. Illumina sRNA-seq 5'衔接头(Illumina,目录号:1000595)
  12. Illumina sRNA-seq PCR引物(Illumina,目录号:1000591,1000592)
  13. 凝胶纯化试剂盒(QIAGEN,目录号:28704)
  14. dNTP(New England Biolabs,目录号:N0447S)
  15. SuperScript II RT(Life Technologies,Invitrogen TM,目录号:18064-022)
  16. Zero Blunt PCR Cloning Kit(Life Technologies,Invitrogen TM,目录号:K2700-40)
  17. 琼脂凝胶(Lonza)

设备

  1. PCR热循环仪
  2. Illumina GA II测序系统
  3. 移液管(20μl,200μl,1000μl)
  4. 磁棒

程序

  1. 使用RNeasy Plant Mini Kit根据制造商的方案(根据制造商的方案,从100mg烟草叶组织获得约60μg高分子量RNA)从植物组织中分离高分子量RNA(长度> 200bp) 。
  2. 使用OligodT Dynabeads根据制造商的方案从10μg总RNA中纯化polyA RNA,并在15μl无RNA酶的水(在该步骤中使用热循环仪和磁棒)中洗脱polyA RNA。
  3. Ligate sRNA 5'衔接子:
    纯化的mRNA
    14μl
    sRNA 5'衔接子(10μM)
    2μl(Illumina sRNA-seq 5'衔接子)
    10x T4 RNA连接酶缓冲液 2μl(*如果不包括ATP,最后加入ATP至1mM)
    T4 RNA连接酶I(10U /μl) 1.5μl
    RNase OUT(40 U /μl) 
    0.5μl
    20℃,6小时
  4. Dynabeads纯化,并根据制造商的方案在15μl无RNA酶的水中洗脱
  5. RNA断裂
    分段缓冲区
    1.6μl(100mM ZnCl 2,100mM Tris-HCl,pH7.0)
    连接mRNA
    14.4μl
    70℃2.5分钟
    使用RNeasy Micro Kit纯化片段化的RNA,纯化后在17μl无RNA酶的水中洗脱RNA
  6. 磷酸酶处理以除去由于断裂产生的3'磷酸酯:
    碎片的RNA
    16μl
    10x磷酸酶缓冲液
    2微升
    南极磷酸酶
    1微升
    RNase OUT(40 U /μl)
    1微升
    37℃,30分钟
    4°C保持
    通过RNeasy Micro Kit纯化RNA,并在15μl无RNA酶的水中洗脱
  7. Ligate sRNA 3'衔接子
    来自步骤6的纯化的RNA
    14.5微升
    10x RNA连接酶缓冲液 2μl(*如果不包括ATP,则添加ATP至1mM最终)
    RNA连接酶1(10Uμl) 2微升
    RNase OUT(40 U /μl)
    1微升
    RNA适配体3'0.5μl
    (10μM,Illumina sRNA-seq 3'衔接头)
    20℃,4小时
  8. 反转录
    制备以下混合物,在70℃加热2分钟并置于冰上。
    衔接子连接的RNA
    4微升
    SRA RT引物
    0.5μl(Illumina sRNA-seq RT引物)
    50 mM dNTP
    0.5μl
    制备以下混合物并加入到上述反应中:
    5x第一链缓冲液
    2微升
    100 mM DTT
    2微升
    RNase OUT(40 U /μl)
    0.25μl
    48℃3分钟,然后加入:
    SuperScript II RT 0.75μl
    44℃60分钟
  9. PCR扩增
    准备以下混合物并加入RT反应:
    Phusion HF 2x mix
    25μl
    引物GX1
    1μl(Illumina sRNA-seq PCR引物)
    引物GX2 
    1μl(Illumina sRNA-seq PCR引物)
    无核酸酶水溶液
    13微升
    运行以下协议:
    1. 98°C 30秒
    2. 30-35个周期:
      98℃10秒
      60°C 30秒
      72℃15秒
    3. 72℃10分钟
    4. 4°C保持
  10. 通过1.5%琼脂糖凝胶运行PCR产物,切割150bp和250bp之间的拖尾区域,并通过凝胶纯化试剂盒纯化,并在25μl洗脱缓冲液中洗脱。
  11. 通过生物分析仪检测文库质量高灵敏度DNA测定法检查大小分布(在此步骤中使用1μl样品,预期生物分析物电泳图谱中约250bp的拖尾区域,参见图1) br />



    图1. dRNA-seq文库的生物分析仪分析上部分是来自生物分析仪运行的电泳图,两条蓝线之间的峰区域代表纯化的dRNA构建体。下面的电泳图显示了通过生物分析仪2100软件对峰区域的分析
  12. 使用Zero Blunt PCR克隆试剂盒克隆几个克隆的文库和序列,以验证来自植物转录物的插入片段的存在。
  13. 使用在Illumina GA II测序系统上运行的小RNA测序来对文库进行测序

致谢

该协议的原理和应用在Li等人(2012)中进行了简要描述。这项工作是由国家科学基金会植物基因组研究计划赠款(DBI-0218166)和联合国支持 国家农业部(CRIS 5335-22000-007-00D)。 作者声明没有利益冲突。

参考文献

  1. Li,F.,Orban,R。和Baker,B。(2012)。 SoMART:用于植物miRNA,tasiRNA和靶基因分析的网络服务器 Plant J 70(5):891-901。
    (当您在出版物中使用此方法时请引用本文)
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How to cite this protocol: Li, F. and Baker, B. (2012). Preparation of cDNA Library for dRNA-seq. Bio-protocol 2(23): e302. DOI: 10.21769/BioProtoc.302; Full Text



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