Aug 01, 2018
Small RNA Sequencing using NEXTflex™ Small RNA-Seq Kit v3 from Bioo Scientific
- 1Girihlet Inc.
- Girihlet Inc Public
Protocol Citation: Kevin D Mohammed, Saboor Hekmaty 2018. Small RNA Sequencing using NEXTflex™ Small RNA-Seq Kit v3 from Bioo Scientific. protocols.io https://dx.doi.org/10.17504/protocols.io.r88d9zw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 31, 2018
Last Modified: August 01, 2018
Protocol Integer ID: 14336
Abstract
GENERAL INFORMATION
Product Overview
The NEXTflex™ Small RNA-Seq Kit v3 can be used to prepare small RNA libraries from total RNA for Illumina-compatible next-generation sequencing. The NEXTflex Small RNA-Seq Kit v3 is designed to greatly reduce formation of adapter-dimer product in small RNA-seq library preparation, allowing completely gel-free library preparation from typical input amounts, or allowing libraries to be created from low input amounts with a PAGE-based size selection of the final library. This kit utilizes patent-pending adapters with randomized ends to greatly reduce sequence bias in small RNA sequencing library construction (1), allowing more accurate identification and quantification of microRNAs, piRNAs, and other small RNAs.
This manual includes protocols for size selection using a gel-free (Step H1) or PAGE-based (Step H2) method. The gel-free protocol described in this manual is recommended for library preparations where a sufficient amount of ~150 bp product and no ~130 bp adapter-dimer product is seen after 16 cycles of PCR, which is typically achieved when using 1μg of total RNA starting material.
Note:
1) This protocol can be adapted for compatibility with sequencing platforms other than Illumina.
The sequences of the adapters (provided in Figure 1) and PCR primers must be modified for the specific sequencing platform.
2) This protocol is specific for version 3 of the NEXTflex™ Small RNA-Seq Kit:
Bioo Scientific, Catalog # 5132-05 (8 reactions), or Catalog # 5132-06 (48 reactions).
Modifications to the protocol may be required for updated versions of the kit.
References
1. Jayaprakash, A.D., et al., Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res, 2011. 39(21): p. e141.
Guidelines
1) Start with 1ug of high quality (determined by Bioanalyzer) total RNA.
2) All steps with beads should be done carefully, pipetting up and down until the sample and beads are clearly mixed and the beads are evenly distributed throughout the sample.
3) Ensure that during bead cleanup steps, the ethanol washes are completely removed from the beads.
4) The NEXTflex Small RNA Sequencing Kit v3 protocol requires 1.5-2 days for completion. Approximate times to complete each step and safe Stopping Points are noted in the protocol; however, careful planning and time management are important for efficient and successful small RNA library preparation. If performing the protocol for the first time, we highly recommend preparing a library with the included microRNA control.
5) There are bead cleanup steps in this protocol in which supernatants must be either be carried over to downstream steps, or in which they are to be discarded. First time users are encouraged to retain all supernatants from bead cleanups in order to troubleshoot failed experiments.
Before start
Determine the concentration and quality of the total RNA sample on an Agilent Bioanalyzer, using Agilent RNA nano or pico kits.
Figure 1: Overview of Small RNA Seq protocol.
Glossary
rAPP- 5' adenylation modification
3ddC- 3' dideoxycytosine
(+) - Sense strand
(-) - Antisense strand
Materials
Materials
NEXTflex™ Small RNA-Seq Kit v3 (Illumina® Compatible) (Bioo Scientific, Catalog # 5132-05 (8 reactions),
or Catalog # 5132-06 (48 reactions).
RNA (1μg total RNA) in up to 10.5 μL Nuclease-free Water (IMPORTANT: see Note below).
Thin wall nuclease-free, low binding PCR tubes.
Nuclease-free, low binding microcentrifuge tubes.
Isopropanol, Molecular Biology Grade (Fisher Scientific, Catalog # BP2618-1)
Absolute ethanol (200 proof), Molecular Biology Grade (Fisher Scientific, Catalog # BP2818-4) for making 80% ethanol, freshly prepared each time.
2, 10, 20, 200 and 1000 μL pipettes.
RNase-free pipette tips.
Microcentrifuge.
Magnetic stand for microcentrifuge tubes (Life Technologies DynaMag™-2, Catalog # 12321D) or similar.
Thermocycler.
Heat block.
Vortex.
Ice.
If conducting PAGE Purification of the final library, these additional items will be needed:
Nuclease-free, low binding 1.7 mL microcentrifuge tubes
0.45μm, 2 mL Spin-X Centrifuge tube (Sigma, Catalog # CLS8162).
Sterile disposable pestles (Fisher Scientific, Catalog # K749521-1500) or similar.
6% TBE PAGE gels (1.0 mm) (Life Technologies Cat # EC6265BOX).
1X TBE Buffer.
Electrophoresis power supply.
Nucleic acid stain such as SYBR Gold (Invitrogen).
UV transilluminator or other visualization tool.
Clean razor or scalpel.
Note
Some total RNA extraction and purification methods may not efficiently isolate small RNAs. Users should verify that their extraction and purification method also isolates small RNAs.
STEP A: NEXTflex™ 3' 4N Adenylated Adapter Ligation
STEP A: NEXTflex™ 3' 4N Adenylated Adapter Ligation
Approximate time to complete: 2.5 hours
Background:
The 3' 4N adenylated adapter is a population of DNA oligonucleotides identical to each other but for the 4 nucleotides at the 5' terminal which are degenerate (4N). This design ameliorates the inherent base-specific biases of RNA ligase, thus allowing more accurate representation of the bonafide relative quatities of a sample's small RNA species in the final sequencing library.
Click on the following link to access the publication demonstrating the effectiveness of this pooling strategy:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241666/
The ligase used in this reaction is a truncated form of T4 RNA ligase 2 (Rnl2), which lacks adenylation activity, but maintains its ligase functionality. Also, unlike its full length counterpart, this truncated ligase cannot ligate the phosphorylated 5' end of DNA or RNA to the 3' end of RNA.
It will ligate only a 5'-end pre-adenylated substrate (DNA or RNA) to the 3' end of an RNA molecule. As such, RNA molecules from the input sample will not be adenylated and thus themselves become substrates for ligation. This prevents circularization or concatenation of input RNA, and specifies the ligation of the adenylated adapter to the 3' end of the input RNA molecules.
Furthermore, the 3' 4N adapters possess at their 3' end a dideoxy nucleotide, thus preventing its ligation to the 5' end of the input RNA, and also to itself.
Figure 2 below shows the final products of this step.
Materials:
Bioo Scientific Supplied
RED CAP - NEXTflex™ 3' 4N Adenylated Adapter, NEXTflex™ 3' Ligation Buffer, NEXTflex™ 3' Ligation Enzyme Mix
WHITE CAP - Nuclease-free Water
User Supplied
RNA (1μg total RNA) in up to 10.5 μL Nuclease-free Water
Nuclease-free, low binding PCR tubes
Thermocycler
Heat block (set to 70°C)
Ice
Figure 2: 3' 4N Adenylated Adapter Ligation overview.
rAPP- 5' adenylation modification
3ddC- 3' dideoxycytosine
(+) - Sense strand
Note
Do not remove NEXTflex™ 3' Ligation Enzyme Mix from -20°C until immediately before use and return to -20°C immediately after use.
For each sample, combine the following reagents on ice in a nuclease-free PCR tube.
| _ μL | RNA (1μg) | |
| _ μL | Nuclease-free Water | |
| 10.5 μL | TOTAL |
Heat at 70°C for 2 minutes in heat block then immediately place on ice.
Incubate on ice for 5 minutes.
Note: Be sure to mix the following reaction until visibly homogenous by pipetting. After mixing, briefly centrifuge to collect contents of tube at the bottom.
For each sample, combine the following reagents on ice in a nuclease-free, low binding PCR tube:
| 10.5 μL | Denatured RNA (from Step 1) | |
| 1 μL | NEXTflex™ 3' 4N Adenylated Adapter | |
| 7 μL | NEXTflex™ 3’ Ligation Buffer | |
| 1.5 μL | NEXTflex™ 3’ Ligation Enzyme Mix | |
| 20 μL | TOTAL |
Mix thoroughly by pipetting, ensuring that tube contents are homogenous.
Briefly centrifuge tubes to collect liquid at bottom of tube.
Incubate at 25°C for 2 hours in a thermocycler with the lid left open.
30 minutes before the end of this incubation, place NEXTflex™ Cleanup Beads (BROWN CAP) at room temperature so that it will be ready for the step in STEP B.
Proceed immediately to Step B: Excess 3' Adapter Removal.
STEP B: Excess 3’ Adapter Removal
STEP B: Excess 3’ Adapter Removal
Approximate time to complete: 1 - 2 hours
Background:
Unligated adapters that are carried downstream in small RNA workflows pose a particular problem, as they form side products known as adapter dimers that tend to dominate the final sequencing reaction.
Steps B and C in this protocol serve to effectively remove free, unligated adapters, thus eliminating the need for tedious PAGE-based isolation of the final desired library from adapter dimers after PCR amplification of the library.
Figure 3 below displays the workflow for this step.
Materials:
Bioo Scientific Supplied
RED CAP - NEXTflex™ Adapter Depletion Solution
YELLOW CAP - Resuspension Buffer
WHITE CAP - Nuclease-free Water
BROWN CAP - NEXTflex™ Cleanup Beads (equilibrate to room temperature for at least 30 minutes; resuspend just prior to use by vortexing for at least 30 seconds).
User Supplied
Isopropanol
80% Ethanol, freshly prepared
Nuclease-free, low binding microcentrifuge tubes
Magnetic Stand for microcentrifuge tubes
*20 μL of NEXTflex™ 3' 4N Adenylated Adapter Ligated RNA (from Step A)
Figure 3: Excess 3’ Adapter Removal and Excess Adapter Inactivation.
3ddC- 3' dideoxycytosine
(+) - Sense strand
To each sample, add 25 μL of NEXTflex™ Adapter Depletion Solution and mix well by pipetting.
Ensure that tube contents are homogenous after mixing.
After mixing, transfer entire sample to a new nuclease-free, low binding microcentrifuge tube.
Add 40 μL of properly resuspended, room temperature equilibrated NEXTflex™ Cleanup Beads to the sample, and mix well by pipetting.
Pipette gently and thoroughly, until beads are homogenously distributed.
Note
Thoroughly resuspend NEXTflex™ Cleanup Beads by vortexing for at least 30 seconds, visually ensuring that beads are no longer settled at the bottom of the bottle.
Immediately add 60 μL of isopropanol and mix well by pipetting.
Pipette gently and thoroughly until beads are homogenously distributed.
Incubate for 5 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Remove supernatant and save for recovery in a new tube (in case final small RNA library generation fails. See 'Note 17' below).
Ensure that all of the supernatant is removed, visually inspecting the tubes.
CONTINUE ONTO STEP 18 OF PROTOCOL WITH THE BEADS IN THE ORIGINAL TUBE.
NOTE 17: Recovery in case of failure to obtain library at end of protocol.
Supernatants to be discarded should be saved in low binding tubes and frozen until the end of the protocol as a failsafe in case library generation fails. Potential pitfalls in the protocol include mistakenly taking the wrong fraction in the bead cleanup/size selection procedures to subsequent steps.
To recover sample, thaw saved supernatants on ice. Add 40ul (for saved supernatants from other downstream steps, be certain to adjust the specifically required volume of beads for those steps) of resuspended NEXTflex™ Cleanup Beads to a low binding tube, magnetize for 5 minutes, remove supernatant while beads are on the magnet.
Add completely thawed saved supernatants to the beads, resuspend well, and proceed with protocol from step 15 above.
Note
Always ensure at steps such as this when supernatants must be removed completely.
Begin aspirating the supernatant from just below its surface and steadily and slowly track the liquid level while aspirating all the way down, making sure to avoid touching the beads.
Additionally, aspirating slowly allows sufficient time for surface tension to drag all the supernatant to the bottom of the tube.
While the tube is on the magnet, add 180 μL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove all of the supernatant.
Repeat this step for a total of 2 ethanol washes.
Remove ethanol completely with samples still engaged on the magnet.
Note
IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods.
Incubate samples for a total of 3 minutes on the magnet.
After the first minute, remove tubes from magnet, briefly centrifuge to collect residual ethanol at bottom of tube.
Replace tubes on the magnet, and incubate for the final 2 minutes.
Once the beads have visually moved to the side of the tube, remove all residual liquid that may have collected at the bottom of the well with a pipette.
Remove sample from magnetic stand and resuspend bead pellet in 22 μL of Resuspension Buffer by pipetting volume up and down. Ensure that beads are completely resuspended.
Spin samples down briefly.
Incubate for 2 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
TRANSFER 20ul OF SUPERNATANT TO A NEW MICROCENTRIFUGE TUBE....
DO NOT DISCARD THIS SUPERNATANT!
Use this supernatant in the next step.
Add 25 μL of NEXTflex Adapter Depletion Solution to the 20ul of supernatant from previous step, and mix well by pipetting.
Add 40 μL of properly resuspended, room temperature equilibrated NEXTflex Cleanup Beads and mix well by pipetting.
Pipette gently and thoroughly, until beads are homogenously distributed.
Immediately add 60 μL of isopropanol and mix well by pipetting.
Pipette gently and thoroughly until beads are homogenously distributed.
Incubate for 5 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Remove supernatant and save for recovery in a new tube (in case final small RNA library generation fails. See 'Note 17' in STEP B-17 above). For recovery, restart procotol at Step 27 after adding thawed saved supernatants to beads.
Ensure that all of the supernatant is removed, visually inspecting the tubes.
CONTINUE ONTO STEP 30 OF PROTOCOL WITH THE BEADS IN THE ORIGINAL TUBE.
While the tube is on the magnet, add 180 μL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove all of the supernatant.
Repeat this step for a total of 2 ethanol washes.
Remove ethanol completely with samples still engaged on the magnet.
Note
IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods.
Incubate samples for a total of 3 minutes on the magnet.
After the first minute, remove tubes from magnet, briefly centrifuge to collect residual ethanol at bottom of tube.
Replace tubes on the magnet, and incubate for the final 2 minutes.
Once the beads have visually moved to the side of the tube, remove all residual liquid that may have collected at the bottom of the well with a pipette.
Remove tubes from magnetic stand and resuspend bead pellet in 13 μL of Nuclease-free water by pipetting. Ensure that beads are completely resuspended.
Incubate for 2 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Transfer 11.5ul of supernatant to a new low binding PCR tube.
Proceed immediately to Step C: Excess Adapter Inactivation.
STOPPING POINT: Alternatively, the procedure may be stopped at this point with samples stored overnight at -20°C. To restart, thaw frozen samples on ice before proceeding to Step C: Excess Adapter Inactivation.
STEP C: Excess Adapter Inactivation
STEP C: Excess Adapter Inactivation
Approximate time to complete: 45 minutes
Materials:
Bioo Scientific Supplied
PINK CAP - NEXTflex™ Adapter Inactivation Buffer, NEXTflex™ Adapter Inactivation Enzyme
User Supplied
Nuclease-free, low binding PCR tubes
Thermocycler
Ice
*11.5 μL of Purified NEXTflex™ 3' 4N Adenylated Adapter Ligated RNA (from Step B)
For each sample, combine the following reagents on ice in a Nuclease-free, low binding PCR tube.
| 11.5 μL | Purified NEXTflex™ 3' 4N Adenylated Adapter Ligated RNA (from Step B) | |
| 2.0 μL | NEXTflex™ Adapter Inactivation Buffer | |
| 0.5 μL | NEXTflex™ Adapter Inactivation Enzyme | |
| 14 μL | TOTAL |
Mix thoroughly by pipetting, ensuring that tube contents are homogenous.
Briefly centrifuge tubes to collect liquid at bottom of tube.
Incubate as follows with lid set to 60°C, but left open on thermocycler (see note below):
| 15 min | 12°C | |
| 20 min | 50°C | |
| hold | 4°C |
Note
- Set thermocycler lid to 60°C, but leave it in the open position during the 12°C incubation, and place it on for the 50°C incubation. Keep samples on ice until PCR block is at 12°C, then transfer the tubes to the thermocycler.
Proceed immediately to Step D: NEXTflex 5' 4N Adapter Ligation.
STEP D: NEXTflex™ 5' 4N Adapter Ligation
STEP D: NEXTflex™ 5' 4N Adapter Ligation
Approximate time to complete: 1.5 hours
Background:
The 5' adapter is an RNA oligonucleotide whose complementary sequence will base pair to the NEXTflex Universal Primer (used in Step G).
As is the case for the 3' adenylated adapter, the 5' adapter also contains a 4 nucleotide region of degeneracy, but at the 3' end of the oligonucleotide, which will form the junction with the 5' end of the RNA species in the reaction.
T4 RNA ligase 1 (Rnl1) in a buffer containing ATP is used to ligate the adapter to the 5' end of the purified 3' 4N adenylated adapter ligation products from STEP A.
Figure 4 below displays the workflow and the final products of this step.
Materials:
Bioo Scientific Supplied
LIGHT PURPLE CAP - NEXTflex™ 5' 4N Adapter, NEXTflex™ 5' Ligation Buffer, NEXTflex™ Ligation Enzyme Mix
User Supplied
Nuclease-free, low binding PCR tubes
Thermocycler
Heat block (set to 70°C)
Ice
*14 μL of Purified NEXTflex™ 3' 4N Adenylated Adapter Ligated RNA (from Step C)
Figure 4: 5' 4N Adapter Ligation overview.
(+) - Sense strand
Note
Do not remove NEXTflex™ NEXTflex™ 5' Ligation Enzyme Mix from -20°C until immediately before use and return to -20°C immediately after use.
Heat 1.5 uL of NEXTflex 5' 4N adapter per reaction at 70°C for 2 minutes in heat block, then immediately place on ice.
Note: Be sure to mix the following reaction until visibly homogenous by pipetting or brief vortexing. For each sample, combine the following reagents on ice in a nuclease-free, low binding PCR tube:
| 14 μL | Purified NEXTflex™ 3' 4N Adenylated Adapter Ligated RNA (from Step C) | |
| 1.5 μL | NEXTflex 5' 4N Adapter | |
| 7.5 μL | NEXTflex™ 5’ Ligation Buffer | |
| 2 μL | NEXTflex™ 5’ Ligation Enzyme Mix | |
| 25 μL | TOTAL |
Mix thoroughly by pipetting, ensuring that the tube contents are homogenous.
Briefly centrifuge tubes to collect liquid at bottom of the tube.
Incubate at 20°C for 1 hour in a thermocycler with the lid left open.
Proceed immediately to Step E: Reverse Transcription - First Strand Synthesis.
STOPPING POINT: Alternatively, the procedure may be stopped at this point with samples stored overnight at -20°C. To restart, thaw frozen samples on ice before proceeding to Step E: Reverse Transcription - First Strand Synthesis.
STEP E: Reverse Transcription-First Strand Synthesis
STEP E: Reverse Transcription-First Strand Synthesis
Approximate time to complete: 1 hour
Background:
The successfully ligated RNAs have (as shown in diagram below), the 5' and 3' adapters attached. The RT primer is complementary to the 3' adapter, and serves as a primer for reverse transcriptase to create the 1st strand cDNA.
Figure 5 below displays the details of the reaction in this step.
Materials:
Bioo Scientific Supplied
BLUE CAP - NEXTflex™ RT Buffer, M-MuLV Reverse Transcriptase
User Supplied
Nuclease-free, low binding PCR tubes
Thermocycler
Ice
*25 μL of 5’ and 3’ NEXTflex™ Adapter Ligated RNA (from Step D)
Figure 5: Reverse transcription of the successfully ligated RNA inserts by a primer complementary to the 3' 4N adapter.
(+) - Sense strand
(-) - Antisense strand
For each sample, combine the following reagents on ice in a Nuclease-free, low binding PCR tube:
| 25 μL | 5' and 3' NEXTflex™ Adapter Ligated RNA (from Step D) | |
| 13 μL | NEXTflex™ RT Buffer | |
| 2 μL | M-MuLV Reverse Transcriptase | |
| 40 μL | TOTAL |
Mix thoroughly by pipetting, ensuring that the tube contents are homogenous.
Briefly centrifuge tubes to collect liquid at bottom of the tube.
Incubate as follows on thermocycler with lid temperature of 100°C.
30 min 42°C
10 min 90°C
Spin tubes down to collect any droplets that may have condensed on lids, and proceed immediately to Step F: Bead Cleanup.
STOPPING POINT: Alternatively, the procedure may be stopped at this point with samples stored overnight at 4°C or up to one week at -20°C. To restart, thaw frozen samples on ice before proceeding to Step F: Bead Cleanup.
STEP F: Bead Cleanup
STEP F: Bead Cleanup
Approximate time to complete: 30 - 45 minutes
Figure 6 below displays an overview of these steps.
Materials:
Bioo Scientific Supplied
RED CAP - NEXTflex™ Adapter Depletion Solution
BROWN CAP - NEXTflex™ Cleanup Beads (equilibrate to room temperature for at least 30 minutes; resuspend just prior to use by vortexing for at least 30 seconds).
WHITE CAP - Nuclease-Free Water
User Supplied
Isopropanol
80% Ethanol, freshly prepared
Magnetic Stand
Nuclease-free, low binding microcentrifuge tubes
*40 μL of First Strand Synthesis product (from Step E)
Figure 6: Excess 5' 4N Adapter Inactivation and Removal.
(-) - Antisense strand
To each sample, add 20 μL of NEXTflex™ Cleanup Beads and mix well by pipetting.
Pipette gently and thoroughly, until beads are homogenously distributed.
Transfer entire sample to a low binding microcentrifuge tube.
Note
Use beads that have been equilibrated to room temperature for at least 30 minutes; resuspend just prior to use by vortexing for at least 30 seconds).
Add 22 μL isopropanol and mix well by pipetting.
Pipette gently and thoroughly until beads are homogenously distributed.
Incubate for 5 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Transfer 70 μL of supernatant to a new low binding microcentrifuge tube.
DO NOT DISCARD SUPERNATANT: this solution contains the cDNA product. Take care to not transfer beads along with clear supernatant.
Ensure that new tubes are removed from magnetic stand.
Add 10 μL NEXTflex™ Adapter Depletion Solution and mix well by pipetting.
Pipette gently and thoroughly, until solution appears homogenous.
Add 20 μL of NEXTflex Cleanup Beads and mix well by pipetting.
Pipette gently and thoroughly, until beads are homogenously distributed.
Note
Resuspend room temperature beads just prior to use by vortexing for at least 30 seconds.
Add 68 μL of isopropanol and mix well by pipetting.
Pipette gently and thoroughly until beads are homogenously distributed.
Incubate for 5 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Remove supernatant and save for recovery in a new tube (in case final small RNA library generation fails. See 'Note 17' in STEP B-17 above).
In case recovery of the saved supernatant from this step (65) is required, ensure that NEXTflex™ Cleanup Bead volume is 20ul (and not 40ul as mentioned in Note 17). Restart procotol at Step 63 after adding thawed saved supernatants to beads.
Ensure that all of the supernatant is removed, visually inspecting the tubes.
CONTINUE ONTO STEP 66 OF PROTOCOL WITH THE BEADS IN THE ORIGINAL TUBE.
While the tube is on the magnet, add 180 μL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove all of the supernatant.
Repeat this step for a total of 2 ethanol washes.
Remove ethanol completely with samples still engaged on the magnet.
Note
IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods.
Incubate samples for a total of 3 minutes on the magnet.
After the first minute, remove tubes from magnet, briefly centrifuge to collect residual ethanol at bottom of tube.
Replace tubes on the magnet, and incubate for the final 2 minutes.
Once the beads have visually moved to the side of the tube, remove all residual liquid that may have collected at the bottom of the well with a pipette.
Remove sample from magnetic stand and resuspend bead pellet in 20 μL Nuclease-free Water by pipetting volume up and down. Ensure that beads are completely resuspended.
Incubate for 2 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Transfer 18 μL of supernatant to a new low binding PCR tube.
Proceed immediately to Step G: PCR Amplification.
STOPPING POINT: Alternatively, the procedure may be stopped at this point with samples stored overnight at -20°C. To restart, thaw frozen samples on ice before proceeding to Step G: PCR Amplification.
STEP G: PCR Amplification
STEP G: PCR Amplification
Approximate time to complete: 40 - 60 minutes
Figure 7 below displays the details of the reaction in this step.
Materials:
Bioo Scientific Supplied
GREEN CAP - NEXTflex™ Barcode Primers, NEXTflex™ Universal Primer, NEXTflex™ Small RNA PCR Master Mix
User Supplied
Low binding PCR tubes
Thermocycler
Ice
*18 μL Purified First Strand Synthesis Product (from Step F)
Figure 7: PCR amplification.
(-) - Antisense strand
For each sample, combine the following reagents on ice in a nuclease-free low bind PCR tubes:
| 18 μL | Purified First Strand Synthesis Product (From Step F) | |
| 1 μL | NEXTflex™ Universal Primer | |
| 1 μL | NEXTflex™ Barcoded Primer (a different barcoded primer should be used for each sample that will be multiplexed for sequencing) | |
| 5 μL | NEXTflex™ Small RNA PCR Master Mix | |
| 25 μL | TOTAL |
Mix thoroughly by pipetting, ensuring that tube contents are homogenous.
Briefly centrifuge tubes to collect liquid at bottom of tube.
Cycle as follows (make sure thermocycler is above 80°C before placing samples on block):
| 2 min | 95°C | ||
| 20 sec | 95°C | 16 cycles. | |
| 30 sec | 60°C | ||
| 15 sec | 72°C | ||
| 2 min | 72°C |
Following PCR, products may be analyzed by Agilent Bioanalyzer HS DNA Assay, TBE-PAGE gel, or similar.
For analysis by Bioanalyzer, we recommend running 1 μL of PCR product diluted 1/4 with nuclease-free water. The Bioanalyzer software may not correctly identify the peak sizes, so it is recommended to also run a library created with miRNA control to help identify the ~150 bp peak. Presence of a strong ~150 bp band indicates a successful library preparation, and absence of a band ~130 bp indicates that gel-free size selection may be used. See Figure 8 below for examples of traces that indicate samples that are amenable to gel-free size selection (A), or that require PAGE-based size selection (B)
Note: A peak of ~65 bp may be present. This peak represents excess PCR primer and will not negatively affect downstream sequencing or quantification by Qubit dsDNA HS Assay.
Figure 8(A): Sample Bioanalyzer HS DNA images of samples that could be size selected with option H1: Gel-Free Size Selection Cleanup
Figure 8(B): Sample Bioanalyzer HS DNA images of samples that could be size selected with H2: PAGE Size Selection and Cleanup.
For analysis by PAGE gel, we recommend mixing 5 μL of PCR product with 1 μL of 6x Loading Dye and running on a 6% TBE-PAGE gel alongside 5 μL of Ready to Load Low Molecular Weight Ladder, and staining with SYBR Gold.
If an ~150bp product is present, and NO detectable 130bp product (adapter dimer), proceed immediately to Step H1: Gel-Free Size Selection Cleanup.
If an ~150bp product is present, and there is an ~130bp product (adapter dimer), proceed immediately to Step H2: Gel Electrophoresis & Nucleic Acid Elution and Purification.
See Figure 9 below for examples of PAGE gel images that indicate samples that are amenable to gel-free size selection (A), or that require PAGE-based size selection (B).
Figure 9: Sample PAGE images of samples that could be size selected with option H1: Gel-Free Size Selection Cleanup (A) or H2: PAGE Size Selection and Cleanup (B).
STOPPING POINT: Alternatively, the procedure may be stopped at this point with samples stored up to one week at -20°C. To restart, thaw frozen samples on ice before proceeding to Step H1: Gel-Free Size Selection Cleanup or Step H2: Gel Electrophoresis & Nucleic Acid Elution and Purification.
STEP H1: Gel-Free Size Selection & Cleanup
STEP H1: Gel-Free Size Selection & Cleanup
Approximate time to complete: 45 minutes
Figure 10 below displays an overview of this section.
Materials:
Bioo Scientific Supplied
WHITE CAP - Nuclease-Free Water
YELLOW CAP - Resuspension Buffer
BROWN CAP - NEXTflex Cleanup Beads (equilibrate to room temperature for at least 30 minutes; resuspend just prior to use by vortexing for at least 30 seconds)
User Supplied
80% Ethanol, freshly prepared
Nuclease-free, low binding microcentrifuge tube
Magnetic Stand
*25 μL of PCR Product (from Step G)
Figure 10: Overview of QC and size selection approaches.
Ensure the volume of all samples is 25 μL. If less, add Nuclease-free Water to bring the entire volume up to 25 uL.
Note
Ensuring that the volume of the PCR reaction is 25ul is critical, as bead-based size selection is dependent on the specific ratio of cleanup beads to sample.
Add 32.5 μL of properly resuspended, room temperature equilibrated NEXTflex™ Cleanup Beads and mix well by pipetting.
Pipette gently and thoroughly, until beads are homogenously distributed.
Incubate for 5 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Transfer 52.5 μL of supernatant to a new nuclease-free, low binding microcentrifuge tube.
DO NOT DISCARD SUPERNATANT, this solution contains the amplified product. Take care to not transfer beads along with clear supernatant.
Ensure that new microcentrifuge tubes with supernatant from previous step are not on the magnet.
Add 30 μL of properly resuspended, room temperature equilibrated NEXTflex Cleanup Beads to each supernatant from previous step, and mix well by pipetting.
Pipette gently and thoroughly, until beads are homogenously distributed.
Incubate for 5 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Remove supernatant and save for recovery in a new tube (in case final small RNA library generation fails. See 'Note 17' in STEP B-17 above).
In case recovery of the saved supernatant from this step (88) is required, ensure that NEXTflex™ Cleanup Bead volume is 30ul (and not 40ul as mentioned in Note 17). Restart procotol at Step 86 after adding thawed saved supernatants to beads.
Be careful to dispose of all the liquid, without disturbing the beads.
CONTINUE ONTO STEP 89 OF PROTOCOL WITH THE BEADS IN THE ORIGINAL TUBE.
While the tube is on the magnet, add 180 μL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove all of the supernatant.
Repeat this step for a total of 2 ethanol washes.
Remove ethanol completely with samples still engaged on the magnet.
Note
IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods.
Incubate samples for a total of 3 minutes on the magnet.
After the first minute, remove tubes from magnet, briefly centrifuge to collect residual ethanol at bottom of tube.
Replace tubes on the magnet, and incubate for the final 2 minutes.
Once the beads have visually moved to the side of the tube, remove all residual liquid that may have collected at the bottom of the well with a pipette.
Remove samples from magnetic stand and resuspend bead pellet in 13.5 μL of Resuspension Buffer by pipetting volume up and down. Ensure that beads are completely resuspended.
Incubate for 2 minutes at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Transfer 12 μL of supernatant to a new low binding microcentrifuge tube.
This is your sequencing library.
Check the size distribution of the final library by Bioanalyzer High Sensitivity DNA Assay (Agilent) and the concentration by Qubit dsDNA HS Assay (Life Technologies).
Figure 11(A) below shows an example of a successfully prepared small RNA library.
Figure 11(A): Sample Bioanalyzer HS DNA traces from libraries created from MCF-7 total RNA using gel-free protocol. Occasionally, a peak of ~65 bp will be seen. This peak represents excess PCR primer and will not negatively affect sequencing or quantification by Qubit dsDNA HS Assay.
(If significant high molecular weight products remain (see Figure 11(B) below for an example trace of a library that would need to be subjected to a second round of size-selection), bring total sample volume to 25 μL with nuclease free water and repeat Step H1).
Figure 11(B): Sample Bioanalyzer HS DNA trace of a library that would benefit from repeating Step H1: Gel-Free Size Selection Cleanup. Elimination of the products ≥180 bp will result in a greater proportion of reads representing small RNAs. Note that repeating step H1 will result in some overall loss.
After second Gel-Free Size Selection, if library traces reflect successful removal of high molecular weight species, proceed to cluster generation for sequencing as per manufacturer's protocol.
STEP H2: PAGE Size Selection & Cleanup
STEP H2: PAGE Size Selection & Cleanup
Note: This option is recommended if an ~150bp product is present, and also an ~130bp adapter dimer after analyzing the PCR products from STEP G (see Figure 8(B) and 9(B).
Note: This step can be replaced with selection by Pippin Prep or Blue Pippin (See Resources tab at http://www.biooscientific.com/SmallRNA)
Approximate time to complete: 3 hours - overnight
Materials:
Bioo Scientific Supplied
YELLOW CAP - Resuspension Buffer
ORANGE CAP - 6X Gel Loading Dye, Ready to Load Low MW Ladder
CLEAR CAP BOTTLE - NEXTflex™ Elution Buffer
BROWN CAP - NEXTflex™ Cleanup Beads
User Supplied
Isopropanol
80% Ethanol
Nuclease-free, low binding 1.7 mL microcentrifuge tubes
Spin-X Centrifuge tube (Sigma)
Sterile disposable pestles (Fisher Cat # K749521-1500 or similar)
Magnetic stand for microcentrifuge tubes (Life Technologies DynaMag™-2 or similar)
6% TBE-PAGE Gel
1X TBE Buffer
Nucleic acid stain such as SYBR Gold (Invitrogen)
UV transilluminator or other visualization tool
Clean razor or scalpel
*25 μL of PCR Product (from Step G)
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 nuclease-free, low binding microcentrifuge tube. Do not cut out the ~130 bp band; this is adapter dimer product. 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 NEXTflex™ 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.
Ensure that NEXTflex Cleanup Beads are placed at room temperature at least 30 minutes before the end of this incubation.
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 Centrifuge tube (Sigma). Cutting the end off of a P1000 tip can help for transfers of larger gel pieces. Centrifuge the Spin-X tube at 16,000 x g for 2 minutes. Dispose of the spin filter.
Ensure that NEXTflex Cleanup Beads are equilibrated to room temperature for at least 30 minutes prior to this step, and that the beads are thoroughly resuspended by vortexing for 30 seconds, with visual confirmation of bead resuspension.
Add to each tube and mix well (Pipette gently and thoroughly, until beads are homogenously distributed):
| NEXTflex Cleanup Beads | 50 μL | |
| Isopropanol | 350 μL |
Incubate at room temperature for 10 minutes. Agitation during this incubation may increase efficiency of recovery.
Pulse spin tubes to collect solution from walls and lid of tube and to pellet beads.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Remove supernatant and save for recovery in a new tube (in case final small RNA library generation fails. See 'Note 17' in STEP B-17 above).
In case recovery of the saved supernatant from this step (114) is required, ensure that NEXTflex™ Cleanup Bead volume is 50ul (and not 40ul as mentioned in Note 17). Restart procotol at Step 111 after adding thawed saved supernatants to beads.
Be careful to dispose of all the liquid, without disturbing the beads.
CONTINUE ONTO STEP 115 OF PROTOCOL WITH THE BEADS IN THE ORIGINAL TUBE.
Add 950 μL 80% ethanol, incubate for 30 seconds, then remove all of the supernatant. Repeat this step for a total of two ethanol washes.
Note
IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods.
Incubate samples for a total of 3 minutes on the magnet.
After the first minute, remove tubes from magnet, briefly centrifuge to collect residual ethanol at bottom of tube.
Replace tubes on the magnet, and incubate for the final 2 minutes.
Once the beads have visually moved to the side of the tube, remove all residual liquid that may have collected at the bottom of the well with a pipette.
Remove samples from magnetic stand 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 at room temperature.
Magnetize samples until solution appears clear (times can range from 2-5 minutes for the clearing of the solution).
Transfer 12 μL of supernatant to a clean nuclease-free, low binding microcentrifuge tube. This is your sequencing library.
Check the size distribution of the final library by Bioanalyzer High Sensitivity DNA Assay (Agilent) and the concentration by Qubit dsDNA HS Assay (Life Technologies).
Proceed to cluster generation for sequencing as per manufacturer's protocol.