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
