Application Note: RNA Analysis
Directional mRNA-Seq
Sample Preparation
Application to prepare directional (strand specific) samples from mRNA
FOR RESEARCH ONLY
ILLUMINA PROPRIETARY
Part # 15018460 Rev. A
October 2010
3
Introduction
4
Sample Prep Workflow
5
Best Practices
7
RNA Input Recommendations
8
Consumables and Equipment
10
Purify mRNA
14
Fragment mRNA
16
Perform Phosphatase Treatment
17
Perform PNK Treatment
18
Purify PNK Treated mRNA
19
Ligate 3' and 5' Adapters
21
Reverse Transcribe and Amplify
24
Enrich DNA Fragments
27
Validate Library
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Application Note: RNA Analysis
3
Introduction
This protocol explains how to prepare directional (strand specific) libraries from mRNA using the Illumina® Small RNA Sample Preparation Kit.
CAUTION
This document describes an experimental application of Illumina technology, which is being shared with the Illumina community as a courtesy. This application is not a standard Illumina kit. Illumina Technical Support scientists and Field Application scientists may not be able to answer questions.
Libraries prepared by this method are compatible with single read flowcells only. Libraries prepared by this method are not compatible with multiplexing. The Illumina Small RNA sequencing primer and Single Read Sequencing Protocol should be used for the sequencing reactions.
Illumina recommends using the Illumina mRNA‐Seq Sample Prep Kit for mRNA purification and fragmentation. Alternative poly‐A selection methods may be used but have not been tested or verified.
4
Sample Prep Workflow
The following figure illustrates the Directional mRNA‐Seq Sample Preparation workflow.
Figure 1 Directional mRNA-Seq Sample Preparation Workflow
Part # 15018460 Rev. A
Application Note: RNA Analysis
5
Best Practices
When preparing mRNA libraries for sequencing, you should always adhere to good molecular biology practices.
RNA Handling
RNA is highly susceptible to degradation by RNAse enzymes. RNAse enzymes are present in cells and tissues, and carried on hands, labware, and dust. They are very stable and difficult to inactivate. For these reasons, it is important to follow best laboratory practices while preparing and handling RNA samples.
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When harvesting total RNA, use a method that quickly disrupts tissue and iso‐
lates and stabilizes RNA.
Wear gloves and use sterile technique at all times.
Reserve a set of pipettes for RNA work. Use sterile RNAse‐free filter pipette tips to prevent cross‐contamination.
Use disposable plasticware that is certified to be RNAse‐free. Illumina recom‐
mends the use of non‐sticky sterile RNAse‐free microcentrifuge tubes. These should not be used for other lab work.
All reagents should be prepared from RNAse‐free components, including ultra pure water.
Store RNA samples at ‐80°C. Avoid extended pauses in the protocol until the RNA is in the form of double‐stranded (ds) DNA.
Use RNAse/DNAse decontamination solution to decontaminate work surfaces and equipment prior to starting this protocol.
Liquid Handling
Good liquid handling measures are essential, particularly when quantifying libraries or diluting concentrated libraries for making clusters.
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Small differences in volumes (±0.5 μl) can sometimes give rise to very large differ‐
ences in cluster numbers (~100,000).
Small volume pipetting can be a source of potential error in protocols that require generation of standard curves, such as PicoGreen assays or qPCR, or those that require small but precise volumes, such as the Agilent BioAnalyzer.
If small volumes are unavoidable, then due diligence should be taken to ensure that pipettes are correctly calibrated.
Ensure that pipettes are not used at the volume extremes of their performance specifications.
Care should be taken, because solutions of high molecular weight dsDNA can be viscous and not evenly dispersed, resulting in aliquot measurements that are not representative of the true concentration of the solution.
6
AMPure Bead Handling
The following indicates the appropriate handling methods when working with Agencourt AMPure XP Beads:
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Prior to use, remove the beads from storage and let stand for at least 30 minutes to bring them to room temperature.
Immediately prior to use, vortex the beads until they are well dispersed. The color of the liquid should appear homogenous.
Set the pipette for the total volume of the beads, up to 200 μl.
After adding the beads to the reaction, mix the solution thoroughly by pipetting up and down 10 times. Change the tips for each sample or when using a multichannel pipette, change the tips after each column.
Let the mixed samples incubate for 15 minutes at room temperature for maximum recovery.
The minimum time required for the beads to be separated from the solution is 5 minutes, but may be longer depending on the volume of the reaction and on the magnetic stand used. Wait at least 5 minutes for the solution to clear before pro‐
ceeding to the next step.
When aspirating the cleared solution from the reaction plate and wash step, it is important to keep the plate on the magnetic stand and do not disturb the sepa‐
rated magnetic beads. Aspirate slowly to prevent the beads from sliding down the sides of the wells and into the pipette tips.
To prevent the loss of beads during purification or the carryover of beads after elution, 5 μl of supernatant are left when the supernatant or eluates are removed from the bead pellet. The remaining liquid does not interfere with subsequent wash steps.
For the wash steps, prepare fresh 80% ethanol. Eighty‐percent ethanol is hygro‐
scopic, therefore, fresh 80% ethanol should be prepared for optimal results.
Be sure to remove all of the ethanol from the bottom of the wells, as it may contain residual contaminants.
Remove the reaction plate from the magnetic stand and let it air‐dry at room tem‐
perature. Allow for the complete evaporation of residual ethanol, as it impacts the performance of the subsequent reactions. If the beads are not completely dried, it is also difficult to remove the eluted DNA in the supernatant without significant bead contamination. Illumina recommends at least 15 minutes drying time, but a longer drying time may be required.
Use a resuspension buffer for DNA elution. When processing 48 or fewer sam‐
ples, gently pipette up and down 10 times, making sure the liquid comes in con‐
tact with the beads and that the beads are resuspended homogeneously.
To maximize DNA recovery during elution, incubate the DNA/bead mix for 2 minutes at room temperature before placing the samples onto the magnet.
Part # 15018460 Rev. A
Application Note: RNA Analysis
7
RNA Input Recommendations
This protocol has been optimized using 1–2 μg total RNA or 50 ng purified mRNA as input. This protocol is designed to start from eukaryotic total RNA and recommends using the poly‐A selection components from the Illumina mRNA‐Seq Sample Preparation Kit. Other mRNA selection methods may be used, as long as the final product is 50–100 ng of full‐length mRNA in 16 μl of nuclease free water. The output mRNA must be of high quality (equivalent to a RIN of 8 or above when run on an Agilent Bioanalyzer RNA chip) and free of inhibitors.
8
Consumables and Equipment
Check to ensure that you have all of the necessary user‐supplied consumables and equipment before proceeding to sample preparation.
Table 1 User-Supplied Consumables
Consumable
Supplier
1.5 ml nuclease‐free, non‐sticky microcentrifuge tubes
General lab supplier
10 mM ATP
Epicentre, part # R109AT or any molecular grade substitute
96‐well V‐bottom Plates
Axygen, catalog # P‐96‐450V‐C
100 mM MgCl2 (can be prepared from 1 M MgCl2 or any molecular grade substitute)
USB, part # 78641 (1 M MgCl2)
80% Ethanol, room temperature
General lab supplier
100% Ethanol, room temperature
General lab supplier
200 μl nuclease‐free, thin‐wall PCR tubes
General lab supplier
Agencourt AMPure XP 60 ml kit
Beckman Coulter Genomics, part # A63881
Antarctic phosphatase with 10X phosphatase buffer
NEB, part # M0289S
MicroAmp Clear Adhesive Film
AB, catalog # 4306311
mRNA‐Seq Sample Prep Kit
Illumina, catalog # RS‐100‐0801
Small RNA Sample Prep Kit
Illumina, catalog # FC‐102‐1009 (8 samples),
or
Illumina, catalog # FC‐102‐1010 (40 samples)
Purified total RNA (1–2 μg) in 10–50 μl volume
User supplied
QIAGEN EB (or substitute 10 mM Tris‐HCI, pH 8.5)
QIAGEN, part # 19086
RNeasy MinElute Cleanup Kit
QIAGEN, part # 74204
SuperScript II Reverse Transcriptase with 100 mM DTT and 5X First Strand Buffer
Invitrogen, part # 18064‐014
T4 Polynucleotide Kinase (PNK) with 10X PNK buffer
NEB, part # M0201S
T4 RNA Ligase 2, truncated with 10X T4 RNL2 truncated reaction buffer
NEB, part # M0242S
Part # 15018460 Rev. A
Application Note: RNA Analysis
9
Table 2 Equipment Checklist
Equipment
Supplier
Benchtop microcentrifuge
General lab supplier
Heat block (2)
General lab supplier
Magnetic stand
General lab supplier
Magnetic stand‐96
Ambion, catalog # AM10027
Room temperature tube rotator
General lab supplier
Thermal cycler
General lab supplier
Vortexer
General lab supplier
10
Purify mRNA
This process purifies the poly‐A containing mRNA molecules using poly‐T oligo‐attached magnetic beads using components from the mRNA‐Seq Sample Prep Kit.
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CAUTION
Allow the beads to fully pellet against the magnetic stand for at least 5 minutes. Remove the supernatant from the beads immediately while the beads are still pelleted against the magnetic stand. Do not allow the pellets to dry.
Illumina-Supplied Consumables
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10 mM Tris Buffer
Bead Binding Buffer
Bead Washing Buffer
Sera‐Mag Magnetic Oligo(dT) Beads
Ultra Pure Water
User-Supplied Consumables
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1–2 μg total RNA
1.5 ml nuclease‐free non‐sticky microcentrifuge tubes
200 μl nuclease‐free, thin‐wall PCR tube
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Application Note: RNA Analysis
11
Procedure
Reference the following workflow diagram while performing the purification procedures.
Figure 2 mRNA Purification Workflow
12
1
Preheat one heat block to 65°C and the other heat block to 80°C.
2
Dilute the total RNA with nuclease‐free ultra pure water to 50 μl in a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube.
3
Heat the sample on a pre‐heated heat block at 65°C for 5 minutes to disrupt the secondary structures, and then place the tube on ice.
4
While the sample is heating, aliquot 15 μl of Sera‐Mag oligo(dT) beads into a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube, then place the tube on the magnetic stand and remove the supernatant.
5
Add 100 μl of Bead Binding Buffer to the tube containing the Sera‐Mag oligo(dT) beads.
6
Remove and discard all of the supernatant from the tube. Take care not to disturb the Sera‐Mag oligo(dT) beads. Gently vortex the tube to mix thoroughly, then centrifuge briefly and place the tube on the magnetic stand.
7
Repeat steps 5 and 6 once for a total of two Bead Binding Buffer washes.
8
Remove the tube from the magnetic stand and resuspend the beads in 50 μl Bead Binding Buffer. Gently vortex the tube to mix thoroughly.
9
Add 50 μl of the total RNA sample from step 3 and mix well.
10 Rotate the tube from step 9 for 5 minutes at room temperature.
11 Place the tube from step 9 on the magnetic stand and remove and discard the supernatant from the tube. Take care not to disturb the beads.
12 Remove the tube from the magnetic stand and wash the beads by resuspending with 200 μl of Bead Washing Buffer. Gently vortex the tube to mix thoroughly.
13 Place the tube on the magnetic stand, then remove and discard the supernatant. Take care not to disturb the beads.
14 Repeat steps 12 and 13 once for a total of two Bead Washing Buffer washes.
15 Add 50 μl of 10 mM Tris‐Buffer to the tube. Gently vortex the tube to mix thoroughly.
16 Aliquot 50 μl of Bead Binding Buffer to a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube.
17 Heat the sample from step 15 on a pre‐heated heat block at 80°C for 2 minutes to elute the mRNA from the beads.
18 Immediately place the tube with the beads on the magnetic stand and transfer all of the supernatant to the tube from step 16 containing the Bead Binding Buffer.
19 Heat the total RNA in the tube without beads on the pre‐heated heat block at 65°C for 5 minutes to disrupt the secondary structures, then place the tube on ice.
20 Wash the beads from step 18 by resuspending with 200 μl of Bead Washing Buffer. Gently vortex the tube to mix thoroughly.
21 Remove and discard the supernatant from the tube. Take care not to disturb the beads.
Part # 15018460 Rev. A
Application Note: RNA Analysis
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22 Repeat steps 20 and 21 once for a total of two Bead Washing Buffer washes.
23 Add 100 μl of mRNA from step 19 to the tube with the beads.
24 Rotate the tube for 5 minutes at room temperature, then place the tube on the magnetic stand.
25 Remove and discard the supernatant from the tube. Take care not to disturb the beads.
26 Wash the beads with 200 μl of Bead Washing Buffer. Gently vortex the tube to mix thoroughly.
27 Remove and discard the supernatant from the tube. Take care not to disturb the beads.
28 Repeat steps 26 and 27 once for a total of two Bead Washing Buffer washes.
29 Add 17 μl of 10 mM Tris‐Buffer to the tube.
30 Heat the tube on the pre‐heated heat block at 80°C for 2 minutes to elute the mRNA from the beads.
31 Immediately place the tube on the magnetic stand and transfer all of the supernatant to a new 200 μl nuclease‐free, thin‐wall PCR tube.
The resulting amount of purified mRNA should be approximately 16 μl.
14
Fragment mRNA
This process fragments the RNA for cDNA synthesis.
Illumina-Supplied Consumables
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5X Fragmentation Buffer
Fragmentation Stop Solution
User-Supplied Consumables
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1.5 ml nuclease‐free, non‐sticky microcentrifuge tubes
80% EtOH
100% EtOH
200 μl nuclease‐free, thin‐wall PCR tube
Nuclease‐free water
The following are provided in the RNeasy MinElute Cleanup Kit:
• RLT
• RNeasy MinElute spin column
• RPE
Template Preparation
1
Pre‐heat the thermal cycler to 94°C.
2
Prepare the following reaction mix on ice in a 200 μl nuclease‐free,thin‐
wall PCR tube:
Reagent
Volume (μl)
5X fragmentation buffer 4
mRNA
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3
Incubate the tube in the pre‐heated thermal cycler at 94°C for exactly 5 minutes, then place the tube on ice.
4
Immediately add 2 μl of Fragmentation Stop Solution to the tube.
Purify Fragmented mRNA
1
Transfer the sample to a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube and add 78 μl nuclease‐free water for a total volume of 100 μl.
2
Add 350 μl RLT buffer. Gently vortex the tube to mix thoroughly, then centrifuge briefly.
Part # 15018460 Rev. A
Application Note: RNA Analysis
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15
Add 675 μl 100% EtOH and invert the tube to mix well.
NOTE
This protocol is a modification to the instructions in the RNeasy MinElute user guide.
4
Immediately load an RNeasy MinElute spin column to a vacuum manifold. Transfer the entire contents of the tube to the spin column.
NOTE
A centrifuge can be used as an alternative to a vacuum manifold. See the RNeasy MinElute user guide.
5
Add 500 μl RPE buffer and vacuum the wash through the column.
6
Add 750 μl 80% EtOH and vacuum the wash through the column.
7
Place the column in a 2 ml collection tube with the lid open.
8
Centrifuge the tube at 13,500 rpm for 5 minutes. Use a 20 μl pipette to remove any residual liquid from the ring of the column.
9
Transfer the column to a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube.
10 Add 17 μl nuclease‐free water to the center of the membrane.
11 Centrifuge the tube at 13,500 rpm for 1 minute to elute the sample.
The total volume should be approximately 16 μl of purified fragmented mRNA.
16
Perform Phosphatase Treatment
This process treats the fragmented mRNA with phosphatase.
Illumina-Supplied Consumables
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RNase Inhibitor
User-Supplied Consumables
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10x phosphatase buffer
200 μl nuclease‐free, thin‐wall PCR tube
Antarctic phosphatase
Template Preparation
1
2
Add the following reagents to a new, 200 μl nuclease‐free, thin‐wall PCR tube and mix well:
Reagent
Volume (μl)
Fragmented mRNA
16
10x phosphatase buffer
2
Antarctic phosphatase
1
RNase Inhibitor
1
Incubate the tube in the thermal cycler as follows:
a 30 minutes at 37°C
b 5 minutes at 65°C
c Hold at 4°C
Part # 15018460 Rev. A
Application Note: RNA Analysis
17
Perform PNK Treatment
This process treats the fragmented mRNA with PNK.
Illumina-Supplied Consumables
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RNase Inhibitor
User-Supplied Consumables
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10 mM ATP
10X PNK buffer
Nuclease‐free water
PNK
Procedure
1
2
Add the following reagents to the 200 μl nuclease‐free,thin‐wall PCR tube containing the 20 μl of phosphatase treated mRNA and mix well:
Reagent
Volume (μl)
Nuclease‐free water
17
10X PNK buffer 5
ATP (10 mM)
5
RNase Inhibitor
1
PNK
2
Incubate the tube in the thermal cycler as follows:
a 60 minutes at 37°C
b Hold at 4°C
18
Purify PNK Treated mRNA
This process uses a column to purify the PNK treated mRNA.
User-Supplied Consumables
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1.5 ml nuclease‐free, non‐sticky microcentrifuge tubes
80% EtOH
100% EtOH
Nuclease‐free water
The following are provided in the RNeasy MinElute Cleanup Kit:
• RLT
• RNeasy MinElute spin column
• RPE
Procedure
1
Transfer the sample to a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube and add 50 μl nuclease‐free water for a total volume of 100 μl.
2
Add 350 μl RLT buffer. Gently vortex the tube to mix thoroughly.
3
Add 675 μl 100% EtOH and invert the tube to mix well.
NOTE
This protocol is a modification to the instructions in the RNeasy MinElute user guide.
4
Immediately load an RNeasy MinElute spin column to a vacuum manifold. Transfer the entire contents of the tube to the spin column.
5
Add 500 μl RPE buffer and vacuum the wash through the column.
6
Add 750 μl 80% EtOH and vacuum the wash through the column.
7
Place the column in a 2 ml collection tube with the lid open.
8
Centrifuge the tube at 13,500 rpm for 5 minutes. Use a 20 μl pipette to remove any residual liquid from the ring of the column.
9
Transfer the column to a new 1.5 ml nuclease‐free, non‐sticky microcentrifuge tube.
10 Add 15 μl nuclease‐free water to the center of the membrane.
11 Centrifuge the tube at 13,500 rpm for 1 minute to elute the sample.
The total volume should be approximately 14 μl of purified fragmented mRNA.
Part # 15018460 Rev. A
Application Note: RNA Analysis
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Ligate 3' and 5' Adapters
This process describes the first ligation reaction of a v1.5 small RNA 3ʹ adapter, followed by the 5ʹ adapter ligation. This process ligates adapters to the 3ʹ and 5ʹ ends of the isolated small RNA.
Illumina-Supplied Consumables
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10X v1.5 sRNA 3ʹ Adapter
SRA 5ʹ Adapter
User-Supplied Consumables
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10 mM ATP
100 mM MgCl2
Nuclease‐free water
T4 RNA Ligase 2, truncated, with 10X T4 RNL2 truncated reaction buffer
200 μl nuclease‐free, thin‐wall PCR tube
Procedure
1
Dilute the 10X v1.5 sRNA 3ʹ (1:10) adapter by mixing 1 μl with 9 μl nuclease‐free water.
NOTE
Prepare fresh dilutions of the adapter with each use.
2
Dilute the 1 M MgCl2(1:10) solution by mixing 100 μl with 900 μl nuclease‐free water.
3
Pre‐heat the PCR thermal cycler to 70°C.
4
Combine the following in sterile, 200 μl nuclease‐free, thin‐wall PCR tube and mix well:
5
Reagent
Volume (μl)
RNA sample
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Diluted v1.5 sRNA 3ʹ adapter
2
Incubate the tube on the pre‐heated thermal cycler at 70°C for 2 minutes, then place the tube on ice.
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6
Add the following regents to the ligation mixture, mix well and centrifuge briefly:
Reagent
Volume (μl)
10X T4 RNL2 truncated reaction buffer 2.5
100 mM MgCl2
2
RNase Inhibitor
1
T4 RNA Ligase2, truncated
3
7
Pre‐heat the PCR thermal cycler to 22°C.
8
Incubate the tube on the pre‐heated thermal cycler at 22°C for 1 hour.
9
With 5 minutes remaining in the incubation, determine the required amount of the SRA 5ʹ Adapter (2 μl per reaction) and aliquot the total amount to a new, 200 μl nuclease‐free, thin‐wall PCR tube.
10 Remove the reaction tube from the thermal cycler and place on ice.
11 Pre‐heat the thermal cycler to 70°C.
12 Place the aliquoted SRA 5ʹ Adapter on the pre‐heated thermal cycler at 70°C for 2 minutes, the place the tube on ice.
13 Pre‐heat the thermal cycler to 20°C.
14 Add the following regents to the ligation mixture from step 7, mix well and centrifuge briefly:
Reagent
Volume (μl)
10 mM ATP
3
Heat Denatured SRA 5ʹ Adapter
2
15 Add 2 μl T4 RNA Ligase to the reaction tube, mix well and centrifuge briefly.
16 Incubate the tube on the pre‐heated thermal cycler at 20°C for 1 hour.
17 Do one of the following:
a It is the preferred method that you proceed to Reverse Transcribe and
Amplify on page 21.
b If necessary, you can store the ligated adapters at 4°C on the thermal cycler overnight if you do not plan to proceed to the next step immediately.
Part # 15018460 Rev. A
Application Note: RNA Analysis
21
Reverse Transcribe and Amplify
This process describes reverse transcription followed by PCR used to create cDNA constructs based on the small RNA ligated with 3ʹ and 5ʹ adapters. This selectively enriches those fragments that have adapter molecules on both ends. The PCR is performed with two primers that anneal to the ends of the adapters.
Illumina-Supplied Consumables
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25 mM dNTP Mix
5X Phusion™ HF Buffer (Finnzymes Oy)
Phusion Polymerase (Finnzymes Oy)
Primer GX1
Primer GX2
RNase Inhibitor
SRA RT Primer
Ultra Pure Water
User-Supplied Consumables
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5ʹ and 3ʹ Adapter‐ligated RNA (4.0 μl)
200 μl nuclease‐free, thin‐wall PCR tubes
SuperScript II Reverse Transcriptase with 100 mM DTT and 5X First Strand Buffer
Prepare Template
1
Pre‐heat the PCR thermal cycler to 70°C.
2
Dilute the SRA RT primer (1:5) by mixing 1 μl with 4 μl nuclease‐free water.
3
Combine the following in a sterile, 200 μl nuclease‐free, thin‐wall PCR tube:
Reagent
Volume (μl)
5ʹ and 3ʹ Ligated RNA
4
Diluted SRA RT Primer 1.0
4
Store the remaining 5ʹ and 3ʹ Ligated RNA at ‐80°C for possible future use.
NOTE
Multiple RT‐PCR reactions can be set up from the same ligation mixture to increase the final yield.
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5
Briefly centrifuge the mixture, then heat the mixture at 70°C on the pre‐heated thermal cycler for 2 minutes, then place the tube on ice.
Dilute the 25 mM dNTP Mix
6
Premix the following reagents in a separate, sterile, 200 μl nuclease‐free, thin‐wall PCR tube. Multiply each volume by the number of samples being prepared. Make 10% extra reagent if you are preparing multiple samples.
Reagent
Volume (μl)
Ultra Pure Water
0.5
25 mM dNTP Mix
0.5
Total Volume per Sample 1
7
Label the tube “12.5 mM dNTP Mix.”
Reverse Transcription
1
Pre‐heat the PCR thermal cycler to 48°C.
2
Premix the following reagents in the order listed in a separate sterile, 200 μl nuclease‐free, thin‐wall PCR tube. The following mix is for one sample. Multiply each volume by the number of samples being prepared. Make 10% extra reagent if you are preparing multiple samples.
Reagent
Volume (μl)
5X First Strand Buffer
2
12.5 mM dNTP Mix
0.5
100 mM DTT
1
RNase Inhibitor
0.5
SuperScript II Reverse Transcriptase
1
Total Volume per Sample
5
3
Add 5 μl of the mix to the iced tube containing the primer‐annealed template material from step 5 of Prepare Template. The total volume should now be 10 μl.
4
Heat the sample on the pre‐heated thermal cycler at 48°C for 3 minutes, then at 44°C for 1 hour.
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Application Note: RNA Analysis
23
Prepare the PCR Master Mix
1
Premix the following reagents in the listed order in a separate sterile, 200 μl nuclease‐free, thin‐wall PCR tube. The following mix is for one sample. Multiply each volume by the number of samples being prepared. Make 10% extra reagent if you are preparing multiple samples.
Reagent
Volume (μl)
Ultra Pure Water
27
5X Phusion HF Buffer
10
Primer GX1
1.0
Primer GX2
1.0
25 mM dNTP Mix
0.5
Phusion DNA Polymerase 0.5
Total Volume per Sample 40
PCR Amplification
1
Add 40 μl of PCR master mix to the sample from step 4 of Reverse
Transcription.
2
Amplify the PCR on the thermal cycler using the following PCR cycling conditions:
NOTE
This process can be programed and saved as the ‘Illumina Small RNA Library Amplification’.
a
b
c
d
30 seconds at 98°C
15 cycles of:
—
10 seconds at 98°C
—
30 seconds at 60°C
—
15 seconds at 72°C
10 minutes at 72°C
Hold at 4°C
SAFE STOPPING POINT
This is a safe stopping point. If you are stopping, store your sample at ‐15° to ‐25°C.
24
Enrich DNA Fragments
User-Supplied Consumables
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1.5 ml nuclease‐free, non‐sticky microcentrifuge tubes
Freshly prepared 80% EtOH
96‐well V‐bottom Plate
AMPure XP Beads
MicroAmp Clear Adhesive Film
QIAGEN EB
First PCR Clean Up
1
Remove the AMPure XP Beads from storage and let stand for at least 30 minutes to bring them to room temperature (Review AMPure Bead
Handling on page 69).
2
Pre‐heat the heat block to 37°C.
3
Seal a new 96‐well v‐bottom plate with a MicroAmp Clear Adhesive Film. Use a razor blade to cut and fold back the seal over the wells to be used in the experiment.
4
Vortex the AMPure XP Beads until they are well dispersed, then add 65 μl of well‐mixed AMPure XP Beads to each unsealed well of the 96‐well v‐bottom plate.
5
Add 50 μl of the PCR reaction mix to each well of the plate containing AMPure XP Beads. Gently pipette the entire volume up and down 10 times to mix thoroughly.
6
Incubate the plate for 8 minutes at room temperature.
7
Place the plate on the magnetic stand‐96 for 5 minutes at room temperature or until the liquid appears completely clear.
8
Remove and discard all of the supernatant from the plate. Take care not to disturb the AMPure XP Beads.
9
Add 200 μl of freshly prepared 80% EtOH to each well without disturbing the beads. Do not remove the plate from the magnetic stand‐96.
10 Incubate the plate for at least 30 seconds at room temperature, then remove and discard the supernatant.
11 Repeat steps 9 and 10 once for a total of two 80% EtOH washes.
12 Re‐seal the open wells and briefly centrifuge the plate to collect all liquid at the bottom of the plate.
13 Place the plate in the magnetic stand‐96 for 1 minute to capture the beads, then remove the entire seal, and then remove the remaining 80% EtOH. Take care not to disturb the beads.
14 Place the plate on the 37°C heat block for 1 minute or until the beads are dry. Small cracks can be observed in the dried bead pellet surface.
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Application Note: RNA Analysis
25
15 Add 30 μl of QIAGEN EB directly to the pellet and let stand for 2 minutes. Gently pipette the entire volume up and down 10 times to mix thoroughly. Ensure that the beads are completely rehydrated and re‐
suspended.
16 Place the plate on the magnetic stand‐96 for 1 minute at room temperature or until the liquid appears completely clear.
Second PCR Clean Up
1
Seal a new 96‐well v‐bottom plate with a MicroAmp Clear Adhesive Film. Use a razor blade to cut and fold back the seal over the wells to be used in the experiment.
2
Vortex the AMPure XP Beads until they are well dispersed, then add 39 μl of well‐mixed AMPure XP Beads to each unsealed well of the 96‐well v‐bottom plate.
3
Transfer 30 μl of liquid from each well of the plate from step 16 of the First
PCR Clean Up to each well of the new plate containing the AMPure XP Beads. Gently pipette the entire volume up and down 10 times to mix thoroughly. 4
Incubate the plate for 8 minutes at room temperature.
5
Place the plate on the magnetic stand‐96 for 5 minutes at room temperature or until the liquid appears completely clear.
6
Remove and discard all of the supernatant from the plate. Take care not to disturb the beads.
7
Add 200 μl of freshly prepared 80% EtOH to each well of the plate without disturbing the beads. Do not remove the plate from the magnetic stand‐96.
8
Incubate the plate for at least 30 seconds at room temperature, then remove and discard the supernatant.
9
Repeat steps 9 and 10 once for a total of two 80% EtOH washes.
10 Re‐seal the open wells and briefly centrifuge the plate to collect all liquid at the bottom of the plate.
11 Place the plate in the magnetic stand‐96 for 1 minute to capture the beads, then re‐open the well seals and remove the remaining 80% EtOH. Take care not to disturb the beads.
12 Place the plate on the 37°C heat block for 1 minute or until the beads are dry. Small cracks can be observed in the dried bead pellet surface.
13 Add 15 μl of QIAGEN EB directly to the pellet and let stand for 2 minutes. Gently pipette the entire volume up and down 10 times to mix thoroughly. Ensure that the beads are completely rehydrated and re‐
suspended.
14 Place the plate on the magnetic stand‐96 for 1 minute at room temperature or until the liquid appears completely clear.
15 Transfer all of the clear supernatant to a new 1.5 ml nuclease‐free non‐sticky microcentrifuge tube.
26
16 Re‐seal the used wells of the plate and you may use the unused wells for future experiments.
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Validate Library
Illumina recommends performing the following quality control analysis on your sample library.
1
Load 1 μl of the resuspended construct on an Agilent Technologies 2100 Bioanalyzer using a DNA specific chip, such as the DNA‐1000 chip.
The product peak should be between 180–200 nucleotides in length.
Figure 3 Final Directional mRNA-Seq Bioanalyzer Trace on a DNA-1000 Chip
2
Check the size, purity, and concentration of the sample.
You can confirm the final product by cloning 1 μl of the product into
Invitrogen Zero Blunt TOPO vector, and sequence using conventional technology.
Illumina, Inc.
9885 Towne Centre Drive
San Diego, CA 92121-1975
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www.illumina.com