Jun 17, 2016

Public workspaceHigh sensitivity (low input) miRNA Illumina library preparation protocol by TailorMix miRNA Sample Preparation Kit V.7

DOI
dx.doi.org/10.17504/protocols.io.e6abhae
  • Karen Yip1,
  • Kelvin Chan1,
  • Danny Lee1
  • 1SeqMatic LLC
Lorraine Lu
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DOI: dx.doi.org/10.17504/protocols.io.e6abhae
External link: http://www.seqmatic.com/wp-content/uploads/2014/07/TM302-Workflow-U14J10002.pdf
Protocol Citation: Karen Yip, Kelvin Chan, Danny Lee 2016. High sensitivity (low input) miRNA Illumina library preparation protocol by TailorMix miRNA Sample Preparation Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.e6abhae
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
Created: June 16, 2016
Last Modified: March 07, 2018
Protocol Integer ID: 2978
Abstract
This protocol is designed for high sensitivity miRNA library generation for the Illumina sequencing platform. Our protocol and the enhanced reagent kit enables the discovery and profiling of small RNAs from a variety of sources including FFPE, exosome, serum, and whole blood. The TailorMix workflow is designed for ease of use, enabling library preparation a single day.

Features:

  • User Friendly Workflow: ­ Libraries can be prepared in a single day.
  • No Additional Reagents Necessary: All reaction enzymes and buffers are provided.
  • Stress Free Gel Purification: TailorCut Gel Extraction Tool Set is included for easy gel excision and purification.
  • Easy to Use: Reagents are supplied as ready-to-use mixtures which improves consistency and reproducibility.
  • Ultra High Sensitivity: Prepare miRNA libraries from any source with as little as 10ng of total RNA input.
Guidelines
Introduction
The TailorMix miRNA Sample Preparation Kit (Version 2) from SeqMatic offers a high sensitivity solution for generating miRNA libraries from low concentration RNA samples. Our kits enable the discovery and profiling of miRNAs from various organisms and tissues via the Illumina sequencing platform. The unique TailorMix reagents and workflow have been developed for simplicity and reproducibility without sacrificing quality or yield.
Figure 1 TailorMix miRNA Sample Preparation V2 Overview


Features
  •  Wide dynamic range
            o Requires as little as 10ng of Total RNA input.
  • User friendly workflow
            o Libraries can be prepared in a single day with less than one hour of hands on time.
  • Comprehensive sample prep kit
            o Most components are supplied as ready-to-use mixtures which improves consistency and reproducibility.
Kit Components
Each TailorMix miRNA Sample Preparation Kit (Version 2) contains two sets of reagents (12 samples), one set of 12 unique barcodes, and one set of gel purification kit.
Set 1: TM302 Reagent Set 1 of 2 (store at -15 °C to -25 °C)
1. Mix A300
2. Mix B300
3. Mix D300
4. Mix E300
5. Mix F300
6. Mix G300
7. Mix H300
Set 2: TM302 Reagent Set 2 of 2 (store at 4 °C)
1. RNA Purification Beads (RPB)
 2. Mix C300
Set 3: PCR Barcode Primers (store at -15 °C to -25 °C)
Set ABarcode Sequence
PCR Primer 
Custom Ladder 
Barcode 1ATCACG
Barcode 2CGATGT
Barcode 3TTAGGC
Barcode 4TGACCA
Barcode 5ACAGTG
Barcode 6GCCAAT
Barcode 7 CAGATC 
Barcode 8 ACTTGA 
Barcode 9 GATCAG 
Barcode 10 TAGCTT 
Barcode 11 GGCTAC 
Barcode 12CTTGTA 
Set BBarcode Sequence
PCR Primer 
Custom Ladder 
Barcode 13 AGTCAA
Barcode 14AGTTCC
Barcode 15ATGTCA
Barcode 16CCGTCC
Barcode 17GTAGAG
Barcode 18GTCCGC
Barcode 19GTGAAA 
Barcode 20GTGGCC 
Barcode 21 GTTTCG 
Barcode 22 CGTACG 
Barcode 23GAGTGG 
Barcode 24GGTAGC 
Set 4: Gel Purification Kit (store at room temperature)
1. Gel Cutter Tools
2. Gel Breaker Tubes
Consumables Preparation
The kit contains all necessary reagents to perform the experiment with the exception of common consumables and instruments. Please make sure all equipment is available before starting this experiment (Table 1).
Table 1 List of Consumables and Equipment
Consumables and Equipment Supplier
1.5 ml nuclease-free microcentrifuge tubesGeneral lab supplier
200 μL, clean, nuclease-free PCR tubesGeneral lab supplier
Nuclease-free waterGeneral lab supplier
TE bufferGeneral lab supplier
Tween-20General lab supplier
Cooler block (optional)SeqMatic, 6388-001
Thermal cyclerGeneral lab supplier
EthanolGeneral lab supplier
Magnetic stand-96 Life Technologies, AM10027
5X TBE buffer General lab supplier 
8% TBE Gels 1.0 mm, 10 WellLIFE Technologies, EC6215BOX
Hi-Density TBE Sample Buffer (5X)LIFE Technologies, LC6678 
 20bp DNA Ladder (Optional) General lab supplier
Ethidium Bromide 10mg/ml  BioRad, 161-0433 
 UV transilluminator General lab supplier 
 SYBR® Gold Nucleic Acid Gel Stain (Optional)LIFE Technologies, S-11494 
Dark reader transilluminator (Optional)  General lab supplier 
 XCell SureLock® Mini-Cell LIFE Technologies, EI0001 
Electrophoresis power supplyGeneral lab supplier 
 Bench top microcentrifuge General lab supplier 
Tube shaker or thermal mixer  General lab supplier 
2100 Bioanalyzer   Agilent Technologies
Agilent High Sensitivity DNA Kit Agilent Technologies, 5067‐4626 
Figure 2 BioAnalyzer High Sensitivity DNA assay of PCR Product (10x diluted) from Human Kidney Tissue Total RNA


Figure 3 Micro RNA Library PCR products on 8% TBE gel


Figure 4 Close up of gel cutting position for miRNA libraries


Figure 5 Close up of gel cutting position for isolating miRNA and other small RNA species


Figure 6 BioAnalyzer High Sensitivity DNA assay of Gel Purified Library from Human Kidney Tissue Total RNA Sample


Best Practices

  • Always wear gloves and use sterile technique.
  • Set up reactions using sterile non-stick nuclease-free tubes.
  • Place samples and reagents on ice or on chilled cooler block at all times and avoid extended pauses.
  • Reagents should be prepared using RNAse-free components
  • Prepare an extra 10% mixture when running multiple samples.
  • Avoid repeated freeze/thaw cycles.

RNA Input

This protocol has been optimized using 10 - 100 ng of purified high quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. You may also use isolated miRNA as the starting material.

Sample Pooling Guidelines

The TailorMix MiRNA Sample Preparation kit is capable of multiplexing up to 96 samples into a single lane of an Illumina flow cell. While processing multiple samples in parallel, use a unique index primer for each sample at the PCR step. Samples can be pooled before or after the library purification step.

Citations & References:

Niu, Jinzhi, et al. “In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.” Insect biochemistry and molecular biology 70 (2016): 127-137.
Materials
MATERIALS
ReagentTailorMix miRNA Sample Preparation Kit V2Catalog #TM302
3’ Adapter Ligation
3’ Adapter Ligation
Thaw Mix C300 from -20°C storage. Allow it to equilibrate to room temperature for a minimum of 30 minutes before use.
Duration00:30:00
Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 25°C if available.
Denature the RNA Sample by assembling the following components in a sterile 200 μL PCR tube on ice:
Reagent Volume (μL)
RNA Sample 6
Mix A300 2
Total 8
Vortex mix thoroughly and incubate at 70°C for 1 minute and then place the tube on ice.
Duration00:01:00
Set up the following 3’ Adapter Ligation reaction on ice:
Reagent Volume (μL)
Denatured RNA mix  from step 4 8
Mix B300 2
Mix C300 6.5
Total 16.5
Note
Note: Mix C300 is a highly viscous reagent. Handle with care and pipette slowly to ensure the correct amount of Mix C300 is dispensed for each reaction.
Vortex mix thoroughly and pulse spin. Incubate at 25°C for 1 hour.
Duration01:00:00
Ligation Product Clean Up
Ligation Product Clean Up
Vortex the TailorMag Purification Beads (TPB) until they are evenly suspended.
Prepare 80% ethanol for rinse step.
Add 30 μL of TPB with each 3’-adapter ligated sample from Step 6. Vortex mix thoroughly and pulse spin.  Iincubate at room temperature for 15 minutes.
Reagent Volume (μL)
3’-adapter ligated sample from Step 6 16.5
TailorMag Purification Beads (TPB) 30
Total 46.5
Duration00:15:00
Note
Note: Do NOT perform strong centrifugation because it will separate TPB from the sample.
Place the sample tube on the magnetic stand at room temperature for 5 minutes.
Duration00:05:00
Carefully remove and discard 40 μL of the supernatant.
Note
Note: Sample recovery may be affected if the TPB pellet is disrupted.
Keep sample tube on the magnetic stand. Gently rinse the TPB pellet with 150 μL of 80% ethanol without disrupting the TPB pellet. Discard the rinse solution.
Note
Tip: Point pipette tip towards opposite direction as the TPB pellet. Gently pipette the 80% ethanol up and down once, then discard the rinse solution.
Air dry sample tube at room temperature.
Note
Note: TailorMag Purification Beads are dried within 5 to 15 minutes at room temperature. Proceed to Step 14 when the appearance of the TPB pellet turns form glossy/shiny (wet) to matte (dry). Sample recovery may be affected if beads are over-dried and appear powdery.
Remove sample tube from the magnetic stand. 
Add 7 μL of nuclease free water to the dried TPB pellet.  
Vortex to resuspend and pulse spin.  
Incubate sample resuspension at room temperature for 2 minutes.
Duration00:02:00
5’ Adapter Ligation
5’ Adapter Ligation
Set up the following 5’ Adapter Ligation reaction on ice:
Reagent Volume (μL)
3’ Adapter Ligated RNA from step 18 7
Mix D300 3
Mix E300 2
Total 12
Gently pipette mix thoroughly and incubate at 25°C for 1 hour and then place the tube on ice.
Duration01:00:00
cDNA Synthesis
cDNA Synthesis
Pre-heat the thermal cycler to 50°C.
Set up the following cDNA Synthesis reaction on ice.
Reagent Volume (μL)
3’ and 5’ Adapter Ligated RNA from Step 16 (contains TPB) 12
Mix F300 2
Mix G300 1
Total 15
Vortex mix thoroughly and pulse spin. Incubate at 50°C for 1 hour and then place the tube on ice.
Duration01:00:00
Note
Safe Stopping Point: First strand cDNA could be stored at -20°C for up to seven days.
PCR Amplification
PCR Amplification
Set up the following PCR reaction in a fresh sterile 200 µl PCR tube on ice:
Reagent Volume (μL)
First strand cDNA from Step 19 (contains TPB) 5
Mix H300 18
PCR Primer 1
Index Primer* 1
Total 25
*Only one of the Index primers is used for each sample.
Note
Note: This protocol has been optimized using 10 - 100 ng of purified high quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization.
An alternative PCR protocol is available for increasing libraries from low yield samples. See the Appendix A in SeqMatic's TailorMix miRNA Sample Preparation 12-reaction Kit (Version 2) for details.
Note: Presence of TPB does not interfere with the enzymatic reaction.
Note: PCR reaction with 5 μL of cDNA generates sufficient amount of libraries for the sequencing run. See Appendix A in SeqMatic's TailorMix miRNA Sample Preparation 12-reaction Kit (Version 2) or PCR set up with the full cDNA volume (15 μL).
Vortex mix thoroughly and pulse spin. Amplify the samples in the thermal cycler using the following PCR cycling conditions:
  • 98°C for 30 seconds
  • 15 cycles of:
  1. 98°C for 5 seconds
  2. 60°C for 15 seconds
  • 72°C for 1 minute
  • 72°C for 5 minutes
  • Hold at 4°C
Note
Safe Stopping Point: PCR products could be stored at -20°C for up to seven days.
PCR yield can be monitored by running an Agilent BioAnalyzer High Sensitivity DNA assay using a dilution of 1 μL of PCR product and 9 μL of nuclease-free water. A typical result shows a distinct peak at approximately 140bp (Figure 2 in Guidelines).
Note
Note: See Appendix B in SeqMatic's TailorMix miRNA Sample Preparation 12-reaction Kit (Version 2) for a more detail description of BioAnalyzer High Sensitivity DNA assay profile of the PCR products.
Library Purification
Library Purification
Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use in gel electrophoresis.
Assemble the gel electrophoresis apparatus.
Mix 2 μL of Custom Ladder with 2 μL of Hi-Density TBE Sample Buffer.
(Optional) Mix 1 μL of 100bp DNA ladder with 1 μL of Hi-Density TBE Sample Buffer.
Add 2.5 μL of Hi-Density TBE Sample Buffer to 25 μL of PCR product and pipet mix thoroughly.
Load 25 μL of the PCR product-Sample Buffer mix into one well in the middle of the 8% PAGE gel. Refer to Figure 3 in Guidelines for an example.
Load 2 μL of the custom ladder and dye mix into the neighboring wells of the PCR products.
Note
Note: Always bracketing each PCR product lane with two custom ladder lanes to ensure precise excision of the miRNA band.
(Optional) Load 2 μL of the 100bp DNA ladder and dye mix into a separate well.
Run the gel for 75 minutes at 145V and immediately remove the gel from the apparatus.
Duration75:00:00
Note
Note: Performance of electrophoresis apparatus varies. Optimization of the setting may be needed for sufficient band separation.
Recover Purified Library
Recover Purified Library
Prepare TE buffer with 0.1% Tween-20.
Reagent Volume (μL)
TE buffer 9,990
Tween-20 10
Total 10,000
Open the gel cassette and stain with 1μg/mL ethidium bromide solution according to the manufacturer’s instructions.
Place the gel on a UV Transilluminator and observe the banding pattern (Figure 3 in Guidelines).
(Alternative) Stain gel with Sybr Gold according to the manufacturer’s instructions and observe the banding pattern on a Dark Reader Transilluminator.
Place the gel breaker tube into a sterile 1.5mL microcentrifuge tube.
The 140bp band represents the highest concentration of micro RNA library. To excise the 140bp band, align the center of the gel cutter tool with the 140 bp band of the custom ladder (Figure 4 in Guidelines).  Press down firmly into the gel and excise the gel fragment.
Note
Note: The 150 bp band represents a combination of micro RNA and other small RNA species (see Appendix B, Q10 if a strong 150bp band is observed). To include the 150bp band in the extraction, align the bottom of the gel cutter tool with the 140bp band of the custom ladder (Figure 5 in Guidelines). Press down firmly into the gel and excise the gel fragment in between the two custom ladder markers.
See Appendix B for a more detail description of the gel bands.
Note
Note: The 150 bp band represents a combination of micro RNA and other small RNA species (see Appendix B in SeqMatic's TailorMix miRNA Sample Preparation 12-reaction Kit (Version 2), Q10 if a strong 150bp band is observed). To include the 150bp band in the extraction, align the bottom of the gel cutter tool with the 140bp band of the custom ladder (Figure 5 in Guidelines). Press down firmly into the gel and excise the gel fragment in between the two custom ladder markers.
See Appendix B in SeqMatic's TailorMix miRNA Sample Preparation 12-reaction Kit (Version 2) for a more detail description of the gel bands.
Insert the gel cutter tool containing the gel slice into the gel breaker tube.
Pulse-spin the gel cutter and gel breaker assembly in a minifuge. Make sure the gel slice is collected in the gel breaker tube.
Remove gel cutter from the assembly and discard. 
Add 30 μL of TE buffer with 0.1% Tween-20 to the gel breaker tube containing the gel slice.
Centrifuge the gel breaker assembly in a bench top centrifuge at maximum speed (approximately 13,000x G) for two minutes at room temperature. Ensure that all of the gel has moved through the holes into the collection tube.
Duration00:02:00
Elute the micro RNA library by shaking the tube at 600 rpm at room temperature overnight.
Duration18:00:00
Note
Note: Do NOT heat up gel buffer mix.
To collect the micro RNA library, spin the gel mix at maximum speed (approximately 13,000x G) for 2 minutes.
Duration00:02:00
With a P10 pipette, gently transfer 20-25 µl of eluate from gel mix to a fresh 1.5ml tube.
Library Validation
Library Validation
Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library. 
Note
Note: Remnant of gel loading dye may appear as a high molecular weight peak on the Bioanalyzer profile. Presence of the dye would not affect performance of the micro RNA library during the sequencing run.
Note: The BioAnalyzer High Sensitivity DNA assay has a 10% deviation on sizing accuracy.
Use 1 μL of resuspended construct from step 47 on a High Sensitivity DNA chip to check the size, purity and concentration of the sample.
Note
Note: Remnant of gel loading dye may appear as a high molecular weight peak on the Bioanalyzer profile. Presence of the dye would not affect performance of the micro RNA library during the sequencing run.
Note: The BioAnalyzer High Sensitivity DNA assay has a 10% deviation on sizing accuracy.