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 ) .
