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February 2014
TailorMix miRNA Sample Preparation Kit
Catalog Numbers: TM100-A and TM100-B
Introduction
The TailorMix miRNA Sample Preparation Kit from SeqMatic is a comprehensive
solution for generating miRNA libraries for the Illumina sequencing platform. Our kits
enable the discovery and profiling of miRNAs from various organisms and tissues. The
unique TailorMix reagents and workflow have been developed for simplicity and
reproducibility without sacrificing quality or yield.
Figure 1 TailorMix MiRNA Sample Preparation Overview
Features

User friendly workflow – libraries can be prepared in a single day with less than one
hour of hands on time.

Comprehensive sample prep kit – most components are supplied as ready-to-use
mixtures which improves consistency and reproducibility.

Wide dynamic range – requires as little as 10 ng of Total RNA input.
29528 Union City Blvd.
Union City, CA 94587
Tel: 510.870.0965
www.seqmatic.com
[email protected]
TailorMix miRNA Sample Preparation Kit v7
February 2014
Components
Each TailorMix miRNA Sample Preparation Kit contains one set of core reagents (12
samples), one set of 12 unique barcodes, and a gel purification kit. The core reagents
and PCR barcode primers should be stored at -15 °C to -25 °C. The kit is designed to
be stable for up to one year after the shipping date.
Set 1: Core Reagents
1.
2.
3.
4.
Mix A
Mix B
Mix C
Mix D
5.
6.
7.
8.
Mix E
Mix F
Mix G
Mix H
Set 2: PCR Barcode Primers
Barcode
Sequence
Catalog # TM100-A
PCR Primer
Custom Ladder
Barcode 1
Barcode 2
Barcode 3
Barcode 4
Barcode 5
Barcode 6
Barcode 7
Barcode 8
Barcode 9
Barcode 10
Barcode 11
Barcode 12
ATCACG
CGATGT
TTAGGC
TGACCA
ACAGTG
GCCAAT
CAGATC
ACTTGA
GATCAG
TAGCTT
GGCTAC
CTTGTA
Catalog # TM100-B
PCR Primer
Custom Ladder
Barcode 13
Barcode 14
Barcode 15
Barcode 16
Barcode 17
Barcode 18
Barcode 19
Barcode 20
Barcode 21
Barcode 22
Barcode 23
Barcode 24
Barcode
Sequence
AGTCAA
AGTTCC
ATGTCA
CCGTCC
GTAGAG
GTCCGC
GTGAAA
GTGGCC
GTTTCG
CGTACG
GAGTGG
GGTAGC
Set 3: Gel Purification Kit
1. Gel Cutter Tool
2. Gel Breaker Tool
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TailorMix miRNA Sample Preparation Kit v7
February 2014
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
1.5 ml nuclease-free microcentrifuge tubes
200 μl, clean, nuclease-free PCR tubes
Nuclease-free water
Supplier
General lab supplier
General lab supplier
General lab supplier
TE buffer
General lab supplier
Tween-20
General lab supplier
Cooler block
IST Engineering, 6388-001
Thermal cycler
General lab supplier
5X TBE buffer
General lab supplier
8% TBE Gels 1.0 mm, 10 Well
LIFE Technologies, EC6215BOX
Hi-Density TBE Sample Buffer (5X)
LIFE Technologies, LC6678
20bp DNA Ladder (Optional)
General lab supplier
SYBR® Gold Nucleic Acid Gel Stain
LIFE Technologies, S-11494
XCell SureLock® Mini-Cell
LIFE Technologies, EI0001
Electrophoresis power supply
General lab supplier
Dark reader transilluminator
Bench top microcentrifuge
Tube shaker or thermal mixer
NanoDrop
2100 Bioanalyzer
High Sensitivity DNA chip
General lab supplier
General lab supplier
General lab supplier
Thermo Scientific
Agilent Technologies
Agilent Technologies, 5067‐4626
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 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 purified 100 ng of high quality human brain
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.
Enabling Accurate NGS
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TailorMix miRNA Sample Preparation Kit v7
February 2014
MiRNA Library Sample Preparation Protocol
3’ Adapter Ligation
Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 25°C if
available.
1.
Denature the RNA by assembling the following components in a sterile 200 μl PCR
tube:
Reagent
Total RNA
Mix A
Total
Volume (μl)
4
2
6
2.
Gently pipette mix thoroughly and incubate at 70°C for 1 minute and then place the
tube on ice.
3.
Set up the following 3’ Adapter Ligation reaction:
Reagent
Denatured RNA and Mix A
from step 2
Mix B
Total
Volume (μl)
6
2
8
4.
Gently pipette mix thoroughly and incubate at 25°C for 1 hour.
5.
With the tube remaining on the thermal cycler, add 1 μl of Mix C directly into each
sample tube.
6.
Gently pipette mix thoroughly and continue to incubate at 25°C for an additional 30
minutes and then place the tube on ice.
5’ Adapter Ligation
7. Set up the following 5’ Adapter Ligation reaction:
Reagent
3’ Ligated RNA from step 6
Mix D
Mix E
Total
8.
Volume (μl)
9
2
1
12
Gently pipette mix thoroughly and incubate at 25°C for 1 hour and then place the
tube on ice.
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TailorMix miRNA Sample Preparation Kit v7
February 2014
cDNA Synthesis
Pre-heat the thermal cycler to 50°C.
9.
Set up the following cDNA Synthesis reaction on ice.
Reagent
3’ and 5’ Ligated RNA from step 8
Mix F
Mix G
Total
10.
Volume (μl)
12
2
1
15
Gently pipette mix thoroughly and incubate at 50°C for 1 hour and then place the
tube on ice.
PCR Amplification
11. Set up the following PCR reaction in a new sterile 200µl PCR tube on ice:
Reagent
cDNA from step 10
Mix H
PCR Primer
Barcode Primer*
Total
Volume (μl)
5
18
1
1
25
*Only one of the barcode primers is used for each sample.
12.
Gently pipette mix thoroughly and amplify the samples in the thermal cycler using
the following PCR cycling conditions:
- 98°C for 30 seconds
- 12 cycles of:
- 98°C for 15 seconds
- 60°C for 15 seconds
- 72°C for 1 minute
- 72°C for 5 minutes
- Hold at 4°C
If the amount of PCR product is insufficient, the number of PCR cycles can be adjusted
up to 15 cycles. See the appendix for details.
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TailorMix miRNA Sample Preparation Kit v7
February 2014
Library Purification
13.
Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use
in gel electrophoresis.
14.
Assemble the gel electrophoresis apparatus.
15.
Mix 2 μl of custom ladder with 2 μl of Hi-Density TBE Sample Buffer.
16.
(Optional) Mix 1 μl of 20bp DNA ladder with 1 μl of Hi-Density TBE Sample Buffer.
17.
Add 2.5 μl of Hi-Density TBE Sample Buffer to 25 μl of PCR product and pipet mix
thoroughly.
18.
Load 25 μl of the PCR product into one well in the middle of the gel. Refer to Figure
3 for an example.
19.
Load 2 μl of the mixed custom ladder into two wells of the 8% PAGE gel separated
by 1-2wells for the PCR products. Bracketing each PCR product lane with two
custom ladder lanes enables a more precise excision of the miRNA band.
20.
(Optional) Load 2 μl of the 20bp DNA ladder and dye mix into a separate well.
21.
Run the gel for 65 minutes at 145V and immediately remove the gel from the
apparatus.
Recover Purified Library
22. Prepare TE buffer with 0.1% Tween-20.
Reagent
TE buffer
Tween-20
Total
Volume (μl)
9,990
10
10,000
23.
Open the gel cassette and stain with Sybr Gold according to the manufacturer’s
instructions.
24.
Place the gel on a Dark Reader Transilluminator and observe the banding pattern
(Figure 3).
25.
Place the gel breaker tube into a sterile 1.5ml microcentrifuge tube or 2.0ml
collection tube.
26.
Gel cutter tool could be used directly or with a P1000 pipette.
Figure 2 Image of gel cutter tool attached to P1000 pipette.
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TailorMix miRNA Sample Preparation Kit v7
27.
February 2014
Align the lower edge of the gel cutter tool with the 140 bp band of the custom ladder
on the sample lane. Press down firmly into the gel and excise the gel fragment.
Figure 3 miRNA Library on TBE gel
20bp
Ladder
Custom
Ladder
MiRNA
Library
Custom
Ladder
20bp
Ladder
500bp
160bp
140bp
Small RNA
Band
28.
Insert the gel cutter tool containing the gel slice into the gel breaker tube.
29.
Briefly spin the gel cutter and gel breaker assembly. Make sure the gel slice is
collected in the gel breaker. Discard gel cutter.
30.
Add 30 μl of TE buffer with 0.1% Tween-20 to the gel breaker tube containing the
gel slice.
31.
Centrifuge the gel breaker assembly in a bench top centrifuge at maximum speed
(approximately 13,000 xg) for two minutes at room temperature. Ensure that all of
the gel has moved through the holes into the collection tube.
32.
Elute the DNA by shaking the tube at 1000 rpm at room temperature for at least one
hour. The tube can be shaken overnight if desired.
33.
To collect the micro RNA library, spin the gel mix at maximum speed
(approximately 13,000 xg) for 2 minutes.
34.
With a P10 pipette, gently remove 20-25 µl eluate from gel mix.
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TailorMix miRNA Sample Preparation Kit v7
February 2014
Library Validation
35.
Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality
control analysis of your sample library. Use 1 μl of resuspended construct from
step 34 on a High Sensitivity DNA chip to check the size, purity and
concentration of the sample.
Figure 4 BioAnalyzer High Sensitivity DNA assayof Gel Purified Library
from a Human Placenta Tissue Total RNA Sample
Small RNA
Library
Sample Pooling
The TailorMix MiRNA Sample Preparation kit is capable of multiplexing up to 24
samples into a single lane of an Illumina flow cell. While processing multiple
samples in parallel, use a unique barcode primer for each sample at the PCR step.
Samples can be pooled before or after the library purification step.
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TailorMix miRNA Sample Preparation Kit v7
February 2014
Appendix: Monitoring PCR Product Yield
PCR yield can be monitored by running an Agilent High Sensitivity DNA or DNA
1000 assay using a dilution of 1 μl of PCR product and 4 μl of nuclease-free water. A
typical result shows a distinct peak at approximately 140bp. If the amount of PCR
product is insufficient, the number of PCR cycles can be adjusted up to 15 cycles.
Figure 5 BioAnalyzer High Sensitivity DNA assay of PCR Product
from a Human Placenta Tissue Total RNA Sample
Small RNA
Library
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