Preparation of cDNA Library for dRNA-seq    

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)