Illumina Small RNA cloning protocol using Random Adapters
Section I: Size selection and Gel purification of RNA sample.
Prepare a 12% polyacrylamide/urea gel
(thickness will depend on the amount of total RNA being run.
As a guide, we use 1.0mm for <20µg,1.5mm for >20µg).
Spike RNA samples with 32P-labeled 19, 24, 28 and/or 33bp oligos (~10,000 counts per oligo, per minute) – depending on the application.
Load the sample(s) and run the gel at constant 10W for 1-1.5 hrs (until the first dye front reaches the middle of the gel).
Add an equal amount of Gel Loading Buffer II (1X), heat samples at 95C for 5 minutes.
Chill on ice for 1 minute.
Carefully grind gel slices by hand using a pestle.
Expose a phosphor-screen over the gel for 10-15 minutes (time will vary depending on radioactivity intensity and imager sensitivity) Add 420µL of 0.4M NaCl.
Spin briefly in a centrifuge at maximum speed.
Flash freeze samples for 1 minute in a bath of EtOH and dry ice.
Transfer eluant to a fresh microcentrifuge tube.
Add 20µg GlycoBlue, mix, and add 2.5 volumes of 100% EtOH Spin at 4C for 30 minutes.
Incubate at -20C for 3-6+ hours.
Air dry for <5 min and resuspend pellet in 13µL DEPC-MilliQ H20 Here, one can insert a gel slice into a dialyzer tube (Novagen, D-Tube Dialyzer Midi, MWCO 3.5 kDA – Cat. No. 71506-3) along with 450ul of water.
Reverse the poles and run for 2 minutes to pull the sample off the dialysis tubing wall.
Position the dialyzer into a gel box, submerged in 1xTAE, such that the dialysis tubing has the current running perpendicularly through it.
Pull out all of the water into a fresh microcentrifuge tube, mix in 20µg GlycoBlue and add 2.5 volumes of 100% EtOH Wash with 70% EtOH and then remove ALL EtOH.
Spin at 4C for 30 minutes Wash with 70% EtOH and then remove ALL EtOH Air dry for <5 min and resuspend pellet in 13µL DEPC-MilliQ H20.
Run at 100V for 18mins.
Set up the ligation mix as described in the cited publication.
Incubate at 37C for 4 hours.
Add 20µl Gel Loading Buffer II.
Section II: 3’ linker ligation.
Section III: Gel purification of 3’ ligated RNA product.
Prepare a 1.0mm, 12% polyacrylamide/urea gel.
Heat inactivate at 95C for 5 minutes.
Heat samples at 95C for 5 minutes.
Chill for 1 minute.
Load the sample(s) and run the gel at constant 10W for 1.5-2 hrs (until the first dye front reaches the bottom of the gel).
Expose a phosphorimager screen over the gel for 30-45 minutes (time will vary) (Figure 2).
Excise the precise band corresponding to the desired size of small RNA (including the ligated radiolabeled oligo(s)) into a microcentrifuge tube.
Spin briefly in a centrifuge at maximum speed.
Carefully grind gel slices using a pestle.
Load 5ng of 32P-labeled Decade Marker as a size marker.
Add 420µL of 0.4M NaCl.
Quickly freeze samples for 1 minute in a bath of EtOH and dry ice.
Incubate overnight at room temperature (RT) with agitation (secured on a vortex).
Spin gel slice homogenate through micropore filter at full speed for 1 minute at RT.
Transfer eluant to a microcentrifuge tube.
Add 20µg GlycoBlue, mix, and add 2.5 volumes of 100% EtOH.
Incubate at -20C for 3-6+ hours.
Spin at 4C for 30 minutes.
Wash with 70% EtOH and then remove ALL EtOH.
Air dry for <5 min and resuspend pellet in 13µL DEPC-MilliQ H20.
Section IV: 5’ linker ligation.
Set up the linker ligation reaction mix as described in the publication.
Incubate at 37C for 2 hours.
Add 20µl Gel Loading Buffer II.
Heat inactivate at 95C for 5 minutes.
Section V: Gel purification of 5’ and 3’ ligated RNA product.
Prepare a 1.0mm, 12% polyacrylamide/urea gel.
Load 2.5ng of 32P-labeled Decade Marker as a size marker.
Heat samples at 95C for 5 minutes.
Chill for 1 minute.
Expose a phosphorimager screen over the gel for 1+ hours (time will vary) (Figure 3).
Load the sample(s) and run the gel at constant 10W for 2+ hrs (until first dye front passes the bottom of the gel).
Spin briefly in a centrifuge at maximum speed.
Excise the precise band corresponding to the desired size of small RNAs (including the ligated radiolabeled oligo(s)) into a microcentrifuge tube.
Spin briefly in a centrifuge at maximum speed.
Add 420µL of 0.4M NaCl.
Quickly freeze samples for 1 minute in a bath of EtOH and dry ice.
Spin gel slice homogenate through micropore filter at full speed for 1 minute at RT.
Transfer eluant to a microcentrifuge tube.
Add 20µg GlycoBlue, mix, and add 2.5 volumes of 100% EtOH.
Incubate at -20oC for 3-6+ hours.
Spin at 4C for 30 minutes.
Carefully grind gel slices using a pestle.
Air dry for <5 min and resuspend pellet in 6.3µL DEPC-MilliQ H20.
Section VI: Reverse transcription.
Incubate at 72C for 2 minutes.
Incubate overnight at room temperature (RT) with agitation (secured on a vortex).
Centrifuge at RT for 1 minute.
Cool on ice for 2 minutes.
Add 8.4µL Reverse Transcriptase (RT) Mix (see publication for details).
Split into two tubes (9µL each).
Add either 1µL Superscript III RT (Invitrogen) (+RT) or 1µL DEPC-MilliQ H20 (-RT control).
Heat to 70C for 15 minutes.
Store at -20C until use.
Incubate at 50C for 1 hour.
Section IX: Quantification of the purified PCR products using a high Sensitivity bioanalyzer.
Section VIII: Ampure Cleanup of amplified cDNA --.
Using a 1.8X ratio, clean up the PCR products following the manufacture’s instructions.
Section VII: PCR amplification of cDNA - see details in publication