Preparation of cDNA Library for dRNA-seq

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The Plant Journal
Jun 2012



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)

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)


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


  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.


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.


  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)


微小RNA(miRNA)是真核生物体中基因表达的普遍存在的调节剂,其指导Argonaute蛋白(AGO)基于序列互补性切割靶mRNA或抑制其翻译。在植物中,miRNA定向切割发生在靶mRNA上,在约5至11个核苷酸(nt)上游至miRNA的5'端结合的位点。 miRNA定向切割产物(降解组)的测序广泛用作验证miRNA介导的调节的生物信息学预测和鉴定已知miRNA的新靶标的方法。在这里,我们描述了使用Illumina GA II测序平台制备高通量测序(dRNA-seq)的降解组RNA互补DNA文库的方案,目前这是最受欢迎和成本效益的。使用这个协议,我们成功地使用三个茄科植物,包括烟草,番茄和马铃薯生成三个dRNA seq图书馆。尽管该协议是用单链适配器开发的,但应该能够通过用多路复用兼容的3'衔接子代替3'衔接子并用索引引物代替PCR引物来产生多重文库。

关键字:NGS, 降解物组, miRNA, siRNA, 目标


  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)


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


  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:
    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) 
  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
    1.6μl(100mM ZnCl 2,100mM Tris-HCl,pH7.0)
    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:
    RNase OUT(40 U /μl)
    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
    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)
    (10μM,Illumina sRNA-seq 3'衔接头)
  8. 反转录
    SRA RT引物
    0.5μl(Illumina sRNA-seq RT引物)
    50 mM dNTP
    100 mM DTT
    RNase OUT(40 U /μl)
    SuperScript II RT 0.75μl
  9. PCR扩增
    Phusion HF 2x mix
    1μl(Illumina sRNA-seq PCR引物)
    1μl(Illumina sRNA-seq PCR引物)
    1. 98°C 30秒
    2. 30-35个周期:
      60°C 30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


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。


  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。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, F. and Baker, B. (2012). Preparation of cDNA Library for dRNA-seq. Bio-protocol 2(23): e302. DOI: 10.21769/BioProtoc.302.