Welcome guest, Login | Register

Home

Preparation of cDNA Library for dRNA-seq

Plant Science > Plant molecular biology > RNA > RNA sequencing
Authors: Feng Li and Barbara Baker
Vol 2, Iss 23, 12/5/2012, 2066 views, 0 Q&A

[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.

Materials and Reagents

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

Equipment

  1. PCR Thermal Cycler
  2. Illumina GA II sequencing system.
  3. Pipette (20 μl, 200 μl, 1000 μl).
  4. Magnetic bar

Procedure

  1. Isolation of high molecular weight RNA (with length > 200bp) from plant tissue using RNeasy Plant Mini Kit according to manufacturer’s protocol (according to the manufacture’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 (IlluminasRNA-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 hour.
  4. Dynabeads purification and elute in 15 μl RNase free water according to manufacture’s protocol
  5. RNA fragmentation
    Fragmentation buffer 1.6 μl (100 mM ZnCl2, 100 mMTris-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 U/μl)        2 μl
    RNase OUT (40 U/μl)          1 μl
    RNA adapter 3’ 0.5 μl         (10 μM, IlluminasRNA-seq 3’ adapter)
    20 °C 4 hour
  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 (IlluminasRNA-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 (IlluminasRNA-seq PCR primer)
    Primer GX2                 1 μl (IlluminasRNA-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
  1. Run the PCR product through a 1.5% Agrose gel (Lonza), cut a smear region between 150 bp and 250 bp and purify by Gel purification kit and elute in 25 μl elution buffer.
  2. Check the library quality by bioanalyzer High sensitivity DNA assay to check the size distribution (one ul sample is used in this step and a smear region centered around 250 bp is expected from the bioanalyzer electrophoresis profile, see Figure 1).




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

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

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, 891-901. (Please cite this paper when use this method in your publications.


How to cite this protocol: Li, F. and Baker, B. (2012). Preparation of cDNA Library for dRNA-seq. Bio-protocol 2(23): e302. http://www.bio-protocol.org/e302



Questions and Comments:

Other protocols by Feng Li
Other protocols by Barbara Baker