TailorMix Small RNA Sample Preparation Kit
Catalog Numbers: TM100-A and TM100-B
Introduction
The TailorMix Small RNA Sample Preparation Kit from SeqMatic is a
comprehensive solution for generating small RNA libraries for the Illumina
sequencing platform, enabling the discovery and profiling of small RNAs from
various organisms and tissues. Our unique TailorMix solution has been
developed for simplicity and reproducibility without sacrificing quality or yield.
This user guide describes the necessary steps to generate libraries for cluster
generation and sequencing. The steps for library generation include adapter
ligation, cDNA synthesis, PCR amplification, and gel purification (Figure 1).
Figure 1 TailorMix Small RNA Sample Preparation Overview
Features
•
User friendly workflow – libraries can be prepared in a single day with under
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 25 ng of Total RNA input.
29528 Union City Blvd.
Union City, CA 94587
Tel: 510.870.0965
www.seqmatic.com
[email protected]
TailorMix Small RNA Sample Preparation Kit
Components
Each TailorMix Small RNA Sample Preparation Kit contains one set of sample
prep reagents (12 samples), one set of 12 unique barcodes, and a set of gel
purification tools. The reagents and should be stored at -15 °C to -25 °C. The kit is
designed to be stable for up to six months after the shipping date.
Set 1: Sample Prep Reagents
1.
2.
3.
4.
5.
6.
7.
8.
9.
3’ Adapter Ligation Mix A
3’ Adapter Ligation Mix B
3’ Adapter Ligation Mix C
5’ Adapter Ligation Mix A
5’ Adapter Ligation Mix B
5’ Adapter Ligation Mix C
cDNA Synthesis Mix A
cDNA Synthesis Mix B
PCR Master Mix
Set 2: Barcode Primers
Barcode
Sequence
Catalog # TM100TM100-A
PCR Primer 1
Barcode 1
Barcode 2
Barcode 3
Barcode 4
Barcode 5
Barcode 6
Barcode 7
Barcode 8
Barcode 9
Barcode 10
Barcode 11
Barcode 12
CGTGAT
ACATCG
GCCTAA
TGGTCA
CACTGT
ATTGGC
GATCTG
TCAAGT
CTGATC
AAGCTA
GTAGCC
TACAAG
Catalog # TM100TM100-B
PCR Primer 1
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
TTGACT
GGAACT
TGACAT
GGACGG
CTCTAC
GCGGAC
TTTCAC
GGCCAC
CGAAAC
CGTACG
CCACTC
GCTACC
Set 3: Gel Purification Tools
1.
2.
3.
4.
Custom DNA Ladder
Gel Cutter Tool
Gel Breaker Tool
5 μm Filter Tubes
2
TailorMix Small RNA Sample Preparation Kit
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
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
Cooler block
IST Engineering, 6388-001
Thermal cycler
General lab supplier
5X TBE buffer
General lab supplier
6% TBE Gels 1.0 mm, 10 Well
LIFE Technologies, EC6265BOX
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 to avoid
contamination.
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 small RNA 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 starting material.
3
TailorMix Small RNA Sample Preparation Kit
Small RNA Library Sample Preparation Protocol
3’ Adapter Ligation
Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 28°C if
available.
1.
Denature the 3’ adapter by assembling the following components in a sterile 200
μl PCR tube:
Reagent
100 ng Total RNA
3’ Adapter Mix A
Total
Volume (μl
( l)
5
2
7
2.
Gently pipette mix thoroughly and incubate at 70°C for 1 minute and then place
the tube on ice.
3.
Set up following the 3’ Adapter Ligation reaction:
Reagent
Denatured 3’ adapter and
RNA
3’ Adapter Mix B
Total
Volume (μl)
( l)
7
2
9
4.
Gently pipette mix thoroughly and incubate at room temperature for 1 hour.
5.
Add 2 μl of 3’ Adapter Ligation Mix C directly into each sample tube.
6.
Gently pipette mix thoroughly and continue to incubate at room temperature for
an additional 15 minutes and then place the tube on ice.
5’ Adapter Ligation
7. Aliquot 2 μl of 5’ Adapter Ligation Mix A into a separate sterile, nuclease-free
200μl PCR tube.
8.
Incubate the tube at 70°C for 1 minute to and then place the tube on ice.
9.
Set up following the 5’ Adapter Ligation reaction:
Reagent
3’ Adapter Ligated RNA
Denatured 5’ adapter
5’ Adapter Ligation Mix B
5’ Adapter Ligation Mix C
Total
10.
Volume (μl)
( l)
9
2
2
2
15
Gently pipette mix thoroughly and incubate at room temperature for 30 minutes
and then place the on ice.
4
TailorMix Small RNA Sample Preparation Kit
cDNA Synthesis
Pre-heat the thermal cycler to 50°C.
11.
Set up following the cDNA Synthesis reaction on a new sterile 200 μl PCR tube on
ice.
Reagent
3’ and 5’ Adapter Ligated RNA
cDNA Synthesis Mix A
cDNA Synthesis Mix B
Total
12.
Volume (μl)
( l)
5.5
3.5
2
11
Gently pipette mix thoroughly and incubate at 50°C for 1 hour and then place the
tube on ice.
PCR Amplification
13. Set up following the PCR reaction:
Reagent
cDNA
PCR Master Mix
PCR Primer 1
Barcode Primer*
Total
Volume (μl)
( l)
11
35
2
2
50
*Only one of the barcode primers is used for each sample.
14.
Gently pipette mix thoroughly and amplify the samples in the thermal cycler
using the following PCR cycling conditions:
- 98°C for 30 seconds
- 13 cycles of:
- 98°C for 2 seconds
- 60°C for 30 seconds
- 72°C for 15 seconds
- 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.
Sample Pooling
The TailorMix Small RNA 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, incorporate the barcode at the PCR step. Samples
can be pooled prior to library purification step.
5
TailorMix Small RNA Sample Preparation Kit
Library Purification
15. Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for
use in gel electrophoresis.
16.
Assemble the gel electrophoresis apparatus.
17.
Mix 2 μl of custom ladder with 2 μl of Hi-Density TBE Sample Buffer.
18.
(Optional) Mix 1 μl of DNA ladder with 1 μl of Hi-Density TBE Sample Buffer.
19.
Add 2 μl of Hi-Density TBE Sample Buffer to PCR product and pipet mix
throroughly.
20.
Load 38 μl of the PCR product into one well in the middle of the gel.
21.
Load 2 μl of the mixed custom ladder into two wells of the 6% PAGE gel separated
by two wells for the PCR product.
22.
(Optional) Load 2 μl of the mixed DNA ladder into a separate well.
23.
Run the gel for 1 hour at 145V and immediately remove the gel from the
apparatus.
Recover Purified Library
24. Open the gel cassette and stain with Sybr Gold according to the manufacturer’s
instructions.
25.
Place the gel on a Dark Reader Transilluminator.
26.
Insert the gel cutter tool to the end of a P1000 pipette.
6
TailorMix Small RNA Sample Preparation Kit
27.
Align the gel cutter tool between the 160 bp and 140 bp band of the custom ladder
on the sample lane. Press down firmly into the gel and excise the gel fragment by
pipetting up.
20bp
Ladder
Custom
Ladder
Small RNA
Library
Custom
Ladder
20bp
Ladder
500bp
160bp
160bp
140bp
40bp
Small RNA
Bands
28.
Expel the gel fragment into the gel breaker tube.
29.
Place the gel breaker tube into a sterile 1.5ml microcentrifuge tube or 2.0ml
collection tube.
30.
Centrifuge the gel breaker tube in a bench top centrifuge at 20,000 xg for two
minutes at room temperature. Ensure that all of the gel has moved through the
holes into the collection tube.
31.
Add 100 μl of nuclease free water or TE buffer to the collection tube containing
the gel debris.
32.
Elute the DNA by shaking the tube at room temperature for at least two hours.
The tube can be shaken overnight if desired.
33.
With a P1000 pipette, transfer the eluate and gel debris into the top of a 5 μm
filter tube.
34.
Centrifuge the filter tube at 600 xg for 10 seconds. The library is now ready for
validation and cluster generation.
7
TailorMix Small RNA Sample Preparation Kit
Library Validation
Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality
control analysis of your sample library.
1. Use 1 μl of resuspended construct on a High Sensitivity DNA chip.
2. Check the size, purity and concentration of the sample.
Figure 2 BioAnalyzer Trace of Gel Purified Library from an
Adult Normal Brain Tissue Total RNA Sample
Small RNA
Library
8
TailorMix Small RNA Sample Preparation Kit
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 two distinct peaks at approximately 140 and 150bp. If the
amount of PCR product is insufficient, the number of PCR cycles can be adjusted
up to 15 cycles. The figure below shows a Bioanalyzer trace from adult lung tissue
total RNA input.
Small RNA
Library
9