be used to saturate all species of sRNAs, allowing recovery of low
abundant sRNAs; (2) it uses spiked
32 P labeled positive controls to
separate sRNAs so that we could track the entire process of sRNA
libraries, allowing precisely identifying the bona fide sRNAs. Alternative method of separation of sRNAs is also provided for the
radioisotope-free laboratories; (3) we have now introduced degenerated adapters (3
0 pre-adenylated oligos) to minimize the bias in
the cloning and sequencing steps; (4) finally, we provide homegenerated scripts to quantify the sRNA reads. Our protocol is
compatible with the prevailing sequencing platforms in the market.
We hope that our protocol can advance plant research community
to clone and sequence sRNAs in a cost-effective and more precise
way.
2 Materials
2.1 Synthesis
of the 5
0
Pre-adenylated
3
0 Adapter
Oligonucleotides
2.1.1 Synthesis
of the Adenosine-5
0 -
phosphoimidazolide (ImpA)
1. Flasks and beakers.
2. Balloon.
3. Nitrogen.
4. Syringes and needles.
5. Rubber septum.
6. Magnetic stirrer and magnetic bars.
7. Corex tubes.
8. Lyophilizer.
9. Centrifuge.
10. 5
0 AMP free acid.
11. Dimethylformamide (DMF).
12. Triphenylphosphine.
13. 2,2
0 -Dipyridyldisulfide.
14. Imidazole (molecular biology grade).
15. Sodium perchlorate.
16. Triethylamine.
17. Acetone.
18. Diethyl ether.
2.1.2 Quality Control of
ImpA
1. UV lamp and silica gel plates.
2. 20 cm  20 cm TLC plate (Silicycle).
3. DMF.
4. Ammonium hydroxide solution.
5. Iso-propanol.
6. Autoclaved double-distilled water.
228
Di Sun et al.
abundant sRNAs; (2) it uses spiked
32 P labeled positive controls to
separate sRNAs so that we could track the entire process of sRNA
libraries, allowing precisely identifying the bona fide sRNAs. Alternative method of separation of sRNAs is also provided for the
radioisotope-free laboratories; (3) we have now introduced degenerated adapters (3
0 pre-adenylated oligos) to minimize the bias in
the cloning and sequencing steps; (4) finally, we provide homegenerated scripts to quantify the sRNA reads. Our protocol is
compatible with the prevailing sequencing platforms in the market.
We hope that our protocol can advance plant research community
to clone and sequence sRNAs in a cost-effective and more precise
way.
2 Materials
2.1 Synthesis
of the 5
0
Pre-adenylated
3
0 Adapter
Oligonucleotides
2.1.1 Synthesis
of the Adenosine-5
0 -
phosphoimidazolide (ImpA)
1. Flasks and beakers.
2. Balloon.
3. Nitrogen.
4. Syringes and needles.
5. Rubber septum.
6. Magnetic stirrer and magnetic bars.
7. Corex tubes.
8. Lyophilizer.
9. Centrifuge.
10. 5
0 AMP free acid.
11. Dimethylformamide (DMF).
12. Triphenylphosphine.
13. 2,2
0 -Dipyridyldisulfide.
14. Imidazole (molecular biology grade).
15. Sodium perchlorate.
16. Triethylamine.
17. Acetone.
18. Diethyl ether.
2.1.2 Quality Control of
ImpA
1. UV lamp and silica gel plates.
2. 20 cm  20 cm TLC plate (Silicycle).
3. DMF.
4. Ammonium hydroxide solution.
5. Iso-propanol.
6. Autoclaved double-distilled water.
228
Di Sun et al.