NEXTflex™ Small RNA Sequencing for Small RNA Starting Material
Vortex and micro centrifuge each component prior to use, to ensure material has not lodged in the cap or the side of the tube.
Add 18 mL of molecular biology-grade water to each bottle of Elution Buffer (10X) to make a 1X Elution Buffer.
Check box on bottle to show water has been added.
Allow 50% PEG to come to room temperature before use.
Inhibitor Incubate at 22°C for 2 hours in a thermocycler.
For ligations to 2’ O-methylated small RNAs, such as those found in plants, incubate at 16°C overnight.
Add 10 µL of Nuclease-free Water to each sample and mix by pipetting.
Add 6 μL of Adapter Depletion Solution and mix well by pipetting.
Add 40 μL of AMPure XP beads and 60 μL of ispropanol and mix well by pipetting.
Incubate for 5 minutes.
Magnetize beads until solution is clear.
Remove and discard supernatant.
Wash #1: Add 180 μL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 μL.
Wash #2: Add 180 μL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 μL.
Incubate sample for 3 minutes.
After one minute, remove all residual liquid that may have collected at the bottom of the well.
Remove plate from magnetic stand and resuspend bead pellet in 22 μL of Resuspenion Buffer by pipetting volume up and down.
Ensure that beads are completely resuspended.
Incubate 2 minutes.
During incubation, add 6 μL of Adapter Depletion Solution to a new, empty well.
Magnetize sample until solution appears clear.
Transfer 20 µL of supernatant to the well containing 6 µL Adapter Depletion Solution and mix well by pipette.
Add 40 µL of AMPure XP beads.
Add 60 µL of isopropanol and mix well by pipetting.
Incubate for 5 minutes.
Magnetize sample until solution appears clear.
Remove and discard supernatant.
Wash #1: Add 180 µL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 µL.
Wash #2: Add 180 µL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 µL.
Incubate sample for 3 minutes.
After one minute, remove all residual liquid that mayhave collected at the bottom of the well.
Remove plate from magnetic stand and resuspend bead pellet in 12 µL of Nuclease-freeWater by pipetting volume up and down.
Ensure that beads are completely resuspended.
Incubate for 2 minutes.
Magnetize sample until solution appears clear.
Transfer 11 µL of supernatant to a new well.
For each reaction, add 1.5 µL of the 5’ NEXTflex™ 4N Adapter and heat at 70°C for 2 minutes, then immediately place on ice.
Incubate at 20°C for 1 hour in a thermocycler.
Add 1 µL NEXTflex™ RT primer to each sample.
Heat at 70°C for 2 minutes then immediately place on ice.
For each sample, add the following components and mix well.
Incubate in a thermocycler at 44°C for 1 hour followed by 90°C for 10 minutes.
The procedure may be safely stopped at this step and samples stored at -20°C.
Add 10 µL of Adapter Depletion Solution to each sample and mix well by pipette.  
Add 40 µL of AMPure XP beads and 90 µL of ispropanol and mix well by pipette.
Incubate for 5 minutes.
Magnetize sample until solution is clear.
Remove and discard supernatant.
Wash #1: Add 180 µL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 µL.
Wash #2: Add 180 µL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 µL.
Incubate sample for 3 minutes.
After one minute, remove any residual liquid that mayhave collected at the bottom of the well.
Remove plate from magnetic stand and resuspend bead pellet in 19 µL of Nuclease-freeWater by pipetting.
Ensure that beads are completely resuspended.
Incubate for 2 minutes.
Magnetize until solution is clear.
Transfer 18 µL of supernatant to a new well.
Add 5 µL of 6X Gel Loading Dye to each PCR product and mix well Load purified PCR products onto a 6% TBE-PAGE gel.
We recommend leaving 1-2 lanes between samples prepared with the same barcode primer to avoid cross contamination.
Samples prepared with different barcodes and that will be sequenced together may be run in adjacent lanes.
In an adjacent lane load 10 µL of Ready to Load Low MW Ladder.
Run the gel with 1X TBE buffer at 200 V until the lower dye band is near the bottom of the gel (0.5-1 cm).
The gel should run for approximately 30 minutes.
Run times may vary depending on individual equipment.
Carefully remove the gel from the glass plates and stain with a nucleic acid stain such as SYBR Gold (Invitrogen) per manufacturer instructions.
Visualize gel bands on a UV transilluminator or other gel documentation instrument.
Using a clean razor cut out the ~150 bp band and place into clean 1.7 mL tube.
Do not cut out the ~130 bp band; this is adapter dimer product.
See Figure 2 in Guidelines for example.
The ladder band at 200 bp is twice as intense as the other bands and can be used for orientation.
Briefly centrifuge the microcentrifuge tube containing the gel slice to collect the gel slice at the bottom of the tube.
Crush the gel slice thoroughly with a disposable pestle.
Leave the pestle in the tube.
Add 300 µL of Elution Buffer to each tube and then remove the pestle, ensuring that as much gel as possible has been washed from the pestle.
Let gel pieces soak at least 2 hours or overnight at room temperature with agitation.
DO NOT incubate longer than overnight.
Pulse spin tubes to collect all eluate from wall and lid.
Carefully transfer the eluate (including crushed gel) to the top of a Spin-X Centrifugetube (Sigma).
Cutting the end off of a P1000 tip can help in transfer of larger gel pieces.
Centrifuge the Spin-X tube at 16,000 x g for 2 minutes.
Dispose of the spin filter.
Add to each tube and mix well*: 50 µL, AMPure XP Beads; 350 µL, Isopropanol. 
Incubate at room temperature for 10 minutes.
Agitation during this incubation may increase efficiency of recovery.
Magnetize sample until solution appears clear.
Wash #1: Carefully remove and discard the supernatant, add 950 µL 80% ethanol, incubate 30 seconds, and remove.
Wash #2: Carefully remove and discard the supernatant, add 950 µL 80% ethanol, incubate 30 seconds, and remove.
Dry sample for 3 minutes.
After one minute, remove all residual liquid that may havecollected at the bottom of the tube.
Remove plate from magnetic rack and resuspend bead pellet in 13 µL of Resuspension Buffer by pipetting volume up and down.
Ensure that beads are completely resuspended and rehydrated.
Incubate for 2 minutes.
Magnetize for 5 minutes or until supernatant appears clear.
Transfer 12 µL of supernatant to a clean 1.7 mL tube.
This is your sequencing library.
Check the size distribution and concentration of the final library by Bioanalyzer HighSensitivity DNA Assay (Agilent).
