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Small RNA Sequencing

Purify small RNA

  • 1. Use FlashPAGE (Ambion) to purify small RNA fraction, directly go to step 17.

  • 2. Alternatively, cast a 1.5mm 15% UREA TBE PAGE, using 5 wells comb.

  • 3. Pre-run the gel at 200V for 15 min.

  • 4. Load 20-100 ug of total RNA with equal volume of formamide loading buffer (95% formamide, 2x TBE, 0.001% bromophenol blue, 0.001% Orange G).

  • 5. Run the gel at 200V until the Orange G dye just runs out.

  • 6. Stain the gel with SYBR gold for 10 min in the dark.

  • 7. Cut the sRNA bands under blue light.

  • 8. Crush the gel by spinning the gel slice through a punctured 0.5 ml epp tube placed in a 2 ml epp tube.

  • 9. Resuspend the gel powder in 1 mL RNase-free TE with 0.3M NaAc pH 5.2, rotate overnight at 4C.

  • 10. Spin down the gel debris and recover the solution containing sRNA.

  • 11. Optional: extract with 2-butanol a few times to remove excessive water if necessary.

  • 12. Add 2 ul glycogen (20mg/ml).

  • 13. Extract with equal volume of chloroform, invert the tube for several times, spin at 14,000rpm, 5 min, 4C.

  • 14. Move the aqueous layer to a new tube, add 3 volumes of 100% EtOH, freeze at -20 C for 0.5 h and spin down the sRNA (14,000rpm, 30mins, 4C).

  • 15. Discard the supernatant, wash the pellet with 500 uL of 75% EtOH, spin the sample at 14,000rpm, 10 mins, 4C, repeat the washing one more time.

  • 16. Air-dry the pellet, dissolve the pellet in 10 – 15 ul DEPC-H2O, roughly determine the concentration of the small RNA (Invitrogen picogreen assay kit).

3' linker ligation

  • 17. Ligate preadenylated 3' linker to small RNA using Truncated T4 RNA ligase 2 (NEB).

  • 18. Incubate the reaction mix at 22C for 5h or 18C overnight.

  • 19. Mix the ligation product with 10 uL of formamide loading buffer.

  • 20. Run Urea-PAGE gel at 200V until the Orange G dye runs out.

  • 21. Stain the gel with SYBR gold in the dark for 10 min.

  • 22. Cut and purify the ligation product (indicated by arrow).


  • 23. Incubate the reaction mix at 22C for 5h or 18C overnight.

  • 24. Mix the ligation product with 10 uL of formamide loading buffer.

  • 25. Run Urea-PAGE gel at 200V until the Orange G dye runs out.

  • 26. Stain the gel with SYBR gold in the dark for 10 min.

  • Cut and purify the ligation product (indicated by arrow).

5' linker ligation

  • 27. Use T4 RNA ligase 1 (Enzymatics) to ligate 5'SR adapter (RNA oligo) to the small RNA with 3' linker. The blocking group in the 3'SR can prevent small RNA self-ligation (circulation) during 5' ligation.

  • 28. Incubate the reaction mix at 25C for 5h or 18C overnight.

Reverse Transcription

  • 29. Add 0.5 uL (2 uM) RT primer to the ligation product, immediately transfer to 60C PCR block for 30 s, incubate on ice and prepare the RT reaction as follow:
    sRNA ligation product with RT primer20μL
    dNTP (10 mM)1.5 uL
    DTT (0.1 M)2 uL
    SuperScriptIII buffer 5x6 uL
    SuperScriptIII0.5 uL

  • 30. Perform RT reaction at 50C for 1 h, heat inactivate at 70C for 15 min.

PCR enrichment

  • 31. Pool all indexed sRNA libraries for PCR:
    RT product (from step 28 or 29)10 uL
    Primer (10 uM)1 uL
    5x Phusion HF Buffer8 uL
    10 mM dNTP1 uL
    H2O19.5 uL
    Phusion II0.5 uL

  • 32. Perform PCR as follow, if the library is over-amplified or not visible, used the remaining RT product and adjust PCR cycle accordingly.

  • 33. Purify sRNA library from the gel, quantify and QC using Agilent bioanalyzer.


Fei Bioinformatics Lab, Boyce Thompson Institute and USDA Robert W. Holley Center