TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide (15049725 v02)

TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide (15049725 v02)
TruSeq® Stranded mRNA
Library Prep for NeoPrep™
Reference Guide
For Research Use Only. Not for use in diagnostic procedures.
Introduction
Additional Resources
RNA Input Recommendations
Pipette and Tip Requirements
Tips and Techniques
Library Prep Workflow
Select Samples and Indexes
Prepare Samples for Loading
Set Up Run and Load Library Card
Unload Libraries
Validate Libraries
Normalize Libraries
Pool Libraries
Supporting Information
Revision History
Technical Assistance
ILLUMINA PROPRIETARY
Catalog # NP-202-9001DOC
Material # 20000945
Document # 15049725 v02
March 2016
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This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the
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The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order
to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read
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© 2016 Illumina, Inc. All rights reserved.
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Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect,
MiniSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA,
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therapeutic or prophylactic purposes in humans or animals.
This protocol explains how to convert the mRNA in total RNA into a library of template
molecules of known strand origin using the reagents provided in the Illumina® TruSeq®
Stranded mRNA Library Prep Kit for NeoPrep™. The resulting libraries are ready for
subsequent cluster generation and sequencing.
The protocol offers:
} Streamlined workflow
} All reagents required for library prep are included
} 30 minutes hands-on time
} Strand information on RNA transcript
} Library capture of both coding RNA and multiple forms of noncoding RNA that are
polyadenylated
} A disposable library card allowing for simultaneous preparation of up to 16 mRNA
samples
} Use of 16 default index adapters, plus 8 alternate index adapters, allowing up to
24-plex pooling with additional NeoPrep runs.
} Compatibility with no indexing or a lower indexing pooling level
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
3
Introduction
Introduction
Additional Resources
Visit the TruSeq Stranded mRNA Library Prep Kit for NeoPrep kit support page on the
Illumina website for documentation, software downloads, training resources, and
information about compatible Illumina products.
The following documentation is available for download from the Illumina website.
4
Resource
Description
Custom Protocol Selector
http://support.illumina.com/custom-protocol-selector.html
A wizard for generating customized end-to-end
documentation that is tailored to the library prep method,
run parameters, and analysis method used for the
sequencing run.
TruSeq Stranded mRNA Library
Prep for NeoPrep Protocol Guide
(document # 15059581)
Provides only protocol instructions.
The protocol guide is intended for experienced users.
TruSeq Stranded mRNA Library
Prep for NeoPrep Checklist
(document # 15068682)
Provides a checklist of the protocol steps.
The checklist is intended for experienced users.
NeoPrep Library Prep System
Guide (document # 15049720)
Provides an overview of instrument components and
software, instructions for performing library prep runs, and
procedures for proper instrument maintenance and
troubleshooting.
Illumina Experiment Manager
Guide (document # 15031335)
and IEM NeoPrep Quick
Reference Card (document #
15061111)
Provide information about creating and editing appropriate
sample sheets for Illumina sequencing systems and analysis
software and record parameters for your sample plate.
BaseSpace help
(help.basespace.illumina.com)
Provides information about the BaseSpace® sequencing data
analysis tool that also enables you to organize samples,
libraries, pools, and sequencing runs in a single
environment.
TruSeq Library Prep Pooling
Guide (document # 15042173)
Provides TruSeq pooling guidelines for preparing libraries
for Illumina sequencing systems that require balanced index
combinations. Review this guide before beginning library
preparation.
Material # 20000945
Document # 15049725 v02
Total RNA Input
}
}
}
}
}
The protocol is optimized for 25–100 ng of total RNA.
} Do not use more than 100 ng of total RNA.
} A lower than specified input amount can result in low yield and increased
duplicates.
The protocol has been tested using 25–100 ng of high-quality universal human
reference total RNA as input.
Determine the quality of the RNA starting material.
} Use an Agilent RNA 6000 Nano Kit or Advanced Analytical Standard Sensitivity
RNA Analysis Kit to determine the quality of your starting material.
} Do not use low quality or degraded RNA with this protocol. Use of degraded RNA
can result in low yield, overrepresentation of the 3' ends of the RNA molecules, or
failure of the protocol.
} Check total RNA integrity following isolation:
} For samples with an RNA Integrity Number (RIN) value ≥ 8, use an Agilent
Technologies 2100 Bioanalyzer.
} For samples with an RNA Quality Number (RQN) value > 8, use an
Advanced Analytical Fragment Analyzer.
} Using RNA with DNA contamination results in an underestimation of the amount
of RNA used.
Include a DNase step with the RNA isolation method to ensure purity and accurate
quantification of the sample.
The following figure shows a Universal Human Reference (UHR) starting RNA
Bioanalyzer trace.
Figure 1 Starting RNA Bioanalyzer Trace
} Alternatively, run a formaldehyde 1% agarose gel and determine the integrity of
RNA upon staining with ethidium bromide.
} High-quality RNA shows a 28S ribosomal RNA (rRNA) band at 4.5 kb with
2X the intensity of the 18S rRNA band at 1.9 kb.
} Both kb determinations are relative to an RNA 6000 ladder.
} The mRNA appears as a smear from 0.5–12 kb.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
5
RNA Input Recommendations
RNA Input Recommendations
Positive Control
Use Agilent Technologies Human UHR total RNA (catalog # 740000) as a positive
control sample for this protocol.
6
Material # 20000945
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Use the following required pipettes and tips. Other pipettes and tips are not supported
and can result in reagents not dispensing properly and run failure.
Table 1 Required User-Supplied Pipettes and Tips Volume
20 µl
200 µl
Use
≤ 20 µl
Product Name
Supplier
Pipet-Lite XLS+ 8-channel LTS, 2 µl to 20 µl Rainin,
catalog # L8-20XLS+
One of the following:
• LTS tips 20 µl. Presterilized. Filter
• Rainin,
catalog # RT-L10F
• Fisher Scientific,
• ART Barrier Pipette Tips 20 µl; 20 µl
catalog # 2749RI
SoftFit-L
21-200 µl Pipet-Lite XLS+ 8-channel LTS,
Rainin,
20 µl to 200 µl
catalog # L8-200XLS+
One of the following:
• LTS tips 200 µl. Presterilized. Filter
• Rainin,
catalog # RT-L200F
• Fisher Scientific,
• ART Barrier Pipette Tips 200 µl; 200 µl
catalog # 2769RI
SoftFit-L
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
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Pipette and Tip Requirements
Pipette and Tip Requirements
Tips and Techniques
Sealing a Plate
}
}
}
Always seal the 96-well plate before the following steps in the protocol:
} Centrifuge
} Thermal cycling
Apply the adhesive seal to cover the plate and seal with a rubber roller.
Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or
semiskirted PCR plates.
Handling the Library Card
}
}
}
To avoid instrument damage, do not place the library card guide on the library card
during library card verification or a run.
Use the library card latch release to load and remove the library card from the library
card stage.
} Do not snap the library card into place.
} Oil and reagents in a used library card can splash out of the card and onto the
instrument.
Hold the used library card level when removing it from the instrument to avoid
spilling its contents.
Library Card Loading Guidelines
}
}
}
}
}
}
}
}
}
}
}
8
Load the library card while it is on the library card stage to avoid spilling or
disturbing the loaded contents.
The NeoPrep Control Software guides you through the steps to set up a run and load
the library card. Use the loading procedures in this guide as a reference.
Perform Set Up Run and Load Library Card preparation and initial software setup
while the sample plate is on the thermal cycler.
Do not open the compartment door during library card verification or a run.
Change your gloves after loading the oil.
Transfer contents from the reagent plate to the corresponding wells on the library
card.
Reference the corresponding colors and well labels on the reagent plate and library
card guides.
Make sure that pipettes are calibrated before beginning. Uncalibrated pipettes can
lead to reagents not dispensing properly, resulting in run failure.
Use the pipettes and tips specified in Pipette and Tip Requirements on page 7 and
Consumables and Equipment on page 29. Other pipettes and tips are not supported and
can result in reagents not dispensing properly and run failure.
Use a multichannel pipette to load reagents, samples, and adapters.
To avoid instrument damage, make sure that the library card guide is removed from
the library card before starting the run.
Material # 20000945
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}
}
}
}
}
}
}
Use proper library card loading techniques and specified loading angles.
Pipette to the first stop to avoid creating bubbles.
Insert pipette tips perpendicular to the well.
Insert the pipette tips to the bottom of the well while dispensing. Do not lift the tips
until the reagents are dispensed completely.
Dispense at an angle by pointing pipette tips under the well label and dotted well
outline on the library card guide.
The pipette loading angle depends on the item being dispensed. The angle is
specified in each step of the control software loading guide and is depicted in the
protocol.
An icon represents the loading angle and the volume is specified on the control
software loading guide. For example,
Icon
Description
Point the pipette tip toward the well label and dotted well outline on the left.
Point the pipette tip perpendicular in the well.
Point the pipette tip toward the well label and dotted well outline on the right.
}
Increase the pipette angle if liquid is not dispensing from the pipette tips.
Handling Samples
}
}
}
}
}
Always track the location of each sample.
Change tips between each sample to avoid cross-contamination.
Do not centrifuge samples before loading.
Touch and keep the pipette tips at bottom of the well while mixing before loading
onto the library card. Use the same tips to load the sample onto the library card.
Some space might be present in the pipette tips during transfer from the sample
plate to the library card.
Collecting Libraries
}
}
}
}
}
}
Unload the library card while it is on the library card stage.
The NeoPrep Control Software guides you through the steps to unload the library
card. Use the procedures in this guide as a reference.
Do not use a 20 µl pipette. It does not fit properly into the library card well.
Insert pipette tips perpendicularly and touch the tips to the bottom of the collection
wells.
Hold down the library card with one hand while removing the tips from the
collection ports to prevent any movement of the card.
An icon represents the required pipette angle, and the volume is specified on the
control software unloading guide. For example,
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
9
Tips and Techniques
Library Card Loading Techniques
}
Inspect each pipette tip to make sure that a blue library droplet is present in the tips
indicated by the control software.
Figure 2 Library Droplet in Pipette Tips
}
}
If a blue library droplet is not visible in each expected pipette tip, do the following:
} Transfer the extracted liquid to the corresponding plate well containing RSB.
} Do not dispense the liquid back into the library card, which can introduce air gaps
and interfere with library extraction.
} Use a single-channel pipette to repeat the transfer 1 time for the wells that did not
contain the blue droplet. Do not attempt the transfer more than 2 times.
Vigorously pipette up and down in RSB to dislodge the blue library droplet from the
pipette tip.
Handling Library Separation Tube Strips
}
}
}
10
Label the tubes to support tracking sample location.
Use the wells of a plate or another device to hold the library separation tube strips
upright.
Do not centrifuge library separation tube strips.
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Library Prep Workflow
Library Prep Workflow
Figure 3 Workflow Diagram
Samples are first manually prepared with RNA Purification Beads 2 (RPB2). Then, oil,
samples, reagents, and adapters are loaded into the library card for a run on the NeoPrep
System. The NeoPrep Control Software guides you through the run setup and library
card loading steps. After the run is complete, the control software guides you through the
process of collecting your libraries from the library card and separating them from the
oil. Validate and normalize collected libraries.
Before proceeding:
} Review Best Practices, available on the Illumina website. See Additional Resources on
page 4.
} Review Supporting Information on page 27. Confirm kit contents and make sure that
you have the requisite equipment and consumables for this protocol.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
11
Select Samples and Indexes
Before beginning library preparation, plan for your NeoPrep System run.
1
Select the samples to use for library prep. Each kit is single-use for 1 NeoPrep System
run and each NeoPrep System run prepares up to 16 samples.
2
Plan the sample locations on the sample plate and the library card.
} Place samples 1–8 in column 1, A–H
} Place samples 9–16 in column 2, A–H
3
Use the default index adapters in the order that they are arranged in the reagent
plate and arrange the samples used with those index adapters accordingly.
Figure 4 TruSeq Stranded mRNA Library Prep for NeoPrep Index Adapters
A
B
C
Adapters A–H (default for samples 1–8)
Adapters I–P (default for samples 9–16)
Adapters Q–X (alternate)
} Indexes are single-use.
} Each sample requires a unique index in a library prep run.
} Each kit includes 24 single-index adapters, allowing for pooling up to 24 samples
with multiple NeoPrep System runs.
} For the index adapter layout, see Reagent Plate Contents on page 27.
} For the index adapter sequences, see Index Adapter Sequences on page 31. The
TruSeq Stranded mRNA Library Prep for NeoPrep adapters are TruSeq LT
single-index adapters.
} Review the planning steps in the TruSeq Library Prep Pooling Guide (document #
15042173) for Illumina sequencing systems that require balanced index
combinations.
4
12
[Optional] Use IEM or BaseSpace Prep tab to record information about your samples
and indexes. The information is used during the run setup.
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This process binds the polyA containing mRNA molecules using oligo-dT attached
magnetic beads in preparation for loading into the library card.
Consumables
}
}
}
}
}
RPB2 (RNA Purification Beads 2)
96-well 0.3 ml PCR plate
Microseal 'B' adhesive seal
Nuclease-free ultrapure water
Total RNA (25–100 ng)
Preparation
1
Remove RPB2 from 2°C to 8°C storage and let stand for at least 15 minutes to bring
to room temperature.
2
Save the following mRNA Denaturation program on a thermal cycler:
} Choose the preheat lid option and set to 100°C
} 65°C for 5 minutes
} 25°C for 5 minutes
} 25°C hold
3
Make sure that pipettes are calibrated before beginning. Uncalibrated pipettes can
lead to reagents not dispensing properly, resulting in run failure.
Procedure
1
Dilute 25–100 ng total RNA with nuclease-free ultrapure water to a final volume of
12.5 µl in each well of a new PCR plate. Pipette to mix. Do not vortex.
2
Vortex RPB2 to resuspend.
3
Add 12.5 µl RPB2 to each well. Pipette to mix.
4
Place on the thermal cycler and run the mRNA Denaturation program.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
13
Prepare Samples for Loading
Prepare Samples for Loading
Set Up Run and Load Library Card
This process describes how to set up a NeoPrep System run, which includes loading oil,
samples, reagents, and adapters into the library card. Load the library card and start the
run within 90 minutes.
The NeoPrep Control Software guides you through the steps to set up a run and load the
library card. Use the procedures in this section as a reference. For more information on
the NeoPrep Control Software, see the NeoPrep Library Prep System Guide (document #
15049720).
Consumables
}
}
}
}
}
}
}
}
Library card
Library card guide
Oil vial
Oil funnel
DMB (Digital Microfluidics Beads)
TruSeq Stranded mRNA - NeoPrep reagent plate
RNase/DNase-free 8-tube strips (2)
[Optional] RSB (Resuspension Buffer)
WARNING
The reagent plate contains hazardous materials. Personal injury can occur through
inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including
eye protection, gloves, and a laboratory coat. Handle the used reagent plate as chemical
waste. Dispose of containers and any unused contents in accordance with the
governmental safety standards for your region. For more information, see the SDS for
this kit at support.illumina.com/sds.html.
About Reagents
Small reagents 9–16 must remain free of RNase contamination. The foil on the reagent
plate is not guaranteed to be RNase-free. Therefore, to prevent the pipette tips from
touching the foil and contaminating these reagents, use a clean 8-tube strip to pierce the
foil on these reagent wells before transferring the reagents.
Preparation
1
Prepare the following consumables:
Item
Reagent plate
Storage
-25°C to -15°C
DMB
2°C to 8°C
RSB
2°C to 8°C
Instructions
Let stand for 15 minutes to bring to room
temperature.
Let stand for 10 minutes to bring to room
temperature.
Let stand for 10 minutes to bring to room
temperature.
Set Up the Run
14
1
Vortex the reagent plate for 3 seconds.
2
Centrifuge at 600 × g for 5 seconds.
If you are not using the reagent plate immediately, set aside on ice.
Material # 20000945
Document # 15049725 v02
Select Prepare Libraries on the NeoPrep System Welcome screen.
4
Do the following and then select Next.
} If running in BaseSpace mode, select a run.
} If running in standalone mode, use the following options to select a protocol:
} Select Select by barcode, and then scan the reagent plate barcode or enter the
reagent plate serial number.
} Select Select by name, and then select TruSeq Stranded mRNA.
5
Configure the run. Select Next.
For more information, see Configure the Run in the NeoPrep Library Prep System Guide
(document # 15049720).
} Configuration options for TruSeq Stranded mRNA are as follows:
Table 2 Configuration Options
Parameter
Default
Sample Count
16
PCR Cycles
15
Options
1–16
12–20
NOTE
Only the default PCR Cycles setting is supported.
6
Review the run and sample information. Select Next.
For more information, see Confirm Run in the NeoPrep Library Prep System Guide
(document # 15049720).
7
Enter the consumable tracking information. Select Next.
For more information, see Track Consumables in the NeoPrep Library Prep System Guide
(document # 15049720).
8
Open the library card compartment door, slide the latch release to the right, and then
place the library card on the library card stage.
WARNING
To avoid instrument damage, make sure that the library card guide is not on the
library card.
9
Close the library card compartment door. Select Verify Library Card. Do not open
the compartment door during library card verification.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
15
Set Up Run and Load Library Card
3
Load the Library Card
1
When library card verification is complete, open the library card compartment door
and place the library card guide on the library card.
Figure 5 Reagent Plate to Library Card Transfer Layout
A
B
C
D
E
F
G
2
Reagent plate
Library card
Oil
Large reagents
Small reagents
Samples
Adapters
Load the entire contents of the oil vial into the library card using the oil funnel. Wait
3 minutes for the oil to drain. Change your gloves after loading the oil.
WARNING
Use the pipette tips specified in Pipette and Tip Requirements on page 7 and
Consumables and Equipment on page 29. Other tips are not supported and can result in
reagents not dispensing properly and run failure.
The loading angle of the pipette depends on the item being dispensed. The angle is
specified in each step of the control software loading guide and is depicted in these
procedures.
3
16
Insert pipette tips to the bottom of the wells of the prepared sample plate. Pipette up
and down 1 time to mix.
Material # 20000945
Document # 15049725 v02
Set Up Run and Load Library Card
4
Transfer 25 µl of prepared samples 1–8.
Figure 6 Loading Samples 1–8
5
Transfer 25 µl of prepared samples 9–16.
Figure 7 Loading Samples 9–16
6
If you are preparing < 16 samples, add 25 µl RSB to empty sample wells.
NOTE
If you are preparing < 9 samples, the NeoPrep Control Software does not provide the
RSB loading instructions for wells 9–16. Add 25 µl RSB to each empty sample well 9–
16.
7
Transfer 125 µl of the large reagents i–iv.
Figure 8 Loading Large Reagents i–iv
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
17
8
Transfer 125 µl of the large reagents v–vii.
Figure 9 Loading Large Reagents v–vii
9
Vortex DMB until well-dispersed. Do not centrifuge DMB.
10 Add 80 µl DMB to the large reagent well viii.
Figure 10 Loading Large Reagent viii
11 Transfer 15 µl of small reagents 1–4, and then 5–8.
Figure 11 Loading Small Reagents 1–4, 5–8
12 For small reagents 9–12:
a
b
Use a clean 8-tube strip to pierce the foil on the reagent wells. Discard the 8-tube
strip.
Transfer 15 µl of each reagent.
Figure 12 Loading Small Reagents 9–12
18
Material # 20000945
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a
b
Use a clean 8-tube strip to pierce the foil on the reagent wells. Discard the 8-tube
strip.
Transfer 15 µl of each reagent.
Figure 13 Loading Small Reagents 13–16
14 Transfer 5 µl of small reagents a–d, and then e–h.
Figure 14 Loading Small Reagents a–d, e–h
NOTE
For steps 15 and 16, if you are not using the default index adapter layout, the control
software specifies which adapter to transfer to each library card well.
15 Transfer 3 µl of adapters A–H.
Figure 15 Loading Adapters A–H
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
19
Set Up Run and Load Library Card
13 For small reagents 13–16:
16 Transfer 3 µl of adapters I–P.
Figure 16 Loading Adapters I–P
NOTE
If you are preparing < 9 samples, the NeoPrep Control Software does not provide the
adapter loading instructions for adapters I–P. Transfer 3 µl of adapters I–P.
17 Remove the library card guide. Keep it for later use during the unloading process.
WARNING
To avoid instrument damage, make sure that the library card guide is removed from
the library card.
18 Close the library card compartment door. Select Start Run. Do not open the
compartment door until the run is complete.
19 When the run is complete, select Next. Libraries can remain at room temperature on
a library card for up to 3 days after a run is complete.
20
Material # 20000945
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This process describes how to collect libraries from the library card, separate libraries
from the oil, and unload the library card from the instrument.
The NeoPrep Control Software guides you through the steps to unload the library card.
Use the procedures in this section as a reference. For more information on the NeoPrep
Control Software, see the NeoPrep Library Prep System Guide (document # 15049720).
Consumables
}
}
}
}
}
RSB (Resuspension Buffer)
Library card guide
Library separation tube strips (2)
96-well 0.3 ml PCR plates (2)
Microseal 'B' adhesive seals
WARNING
The used library card contains hazardous materials. Personal injury can occur through
inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including
eye protection, gloves, and a laboratory coat. Handle the used library card as chemical
waste. Dispose of containers and any unused contents in accordance with the
governmental safety standards for your region. For more information, see the SDS for
this kit at support.illumina.com/sds.html.
Preparation
1
Remove RSB from 2°C to 8°C storage and bring to room temperature.
2
Label wells of 2 new PCR plates 1–16.
3
Label tubes of a library separation tube strip 1–8 and another library separation tube
strip 9–16.
Procedure
1
Add 10 µl RSB to each well of a new PCR plate labeled 1–16.
2
Open the library card compartment door and place the library card guide on the
library card.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
21
Unload Libraries
Unload Libraries
3
Use a 200 µl pipette to transfer 20 µl from library card collection wells 1L–8L, and
then 9L–16L to corresponding wells 1–16 of the plate. Pipette to mix.
Figure 17 Library Card Collection Wells
A
B
Collection wells 1L–8L
Collection wells 9L–16L
4
Centrifuge briefly.
5
Transfer the entire volume from plate wells 1–8, and then 9–16 to the center indent
in the membrane of the corresponding library separation tubes 1–16.
6
Let stand for 10 seconds while the oil is absorbed in the tubes.
7
Transfer the entire volume from library separation tubes 1–8, and then 9–16 to the
corresponding wells 1–16 of a new PCR plate.
8
Remove the library card and library card guide from the library card stage.
9
Discard the library card in accordance with applicable standards.
10 Close the library card compartment door, and then select Home.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
22
Material # 20000945
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Illumina recommends performing the following procedures for quality control analysis
on your sample library and quantification of the DNA library templates.
Consumables
}
}
One of the following for quantification:
} KAPA Library Quantification Kit
} Fluorometric quantification with dsDNA binding dye reagents
One of the following for quality check:
} Agilent DNA 7500 Kit
} Agilent High Sensitivity DNA Kit
Quantify Libraries
To achieve the highest-quality data on Illumina sequencing platforms, it is important to
create optimum cluster densities across every lane of the flow cell. Optimizing cluster
densities requires accurate quantification of DNA libraries.
1
Quantify the libraries using qPCR according to the Illumina Sequencing Library qPCR
Quantification Guide (document # 11322363).
Check Library Quality
1
If using a Standard Sensitivity NGS Fragment Analysis Kit on an Advanced
Analytical Fragment Analyzer:
a
b
Dilute the DNA library 1:1 with RSB.
Run 1 µl diluted DNA library.
2
If using a DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer, run 1 µl
undiluted DNA library.
3
Check the size and purity of the sample. Expect the final product to be a band at
~300 bp.
Figure 18 Example Library Size Distribution with Peak at ~300 bp (Before Normalization)
NOTE
A blue dye is used in the TruSeq Stranded mRNA Library Prep for NeoPrep reagents to
aid in loading and collection. The dye appears as a characteristic peak at 200–250 bp and is
not indicative of issues with the final library. Figure 19 shows the distribution of only the
blue dye.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
23
Validate Libraries
Validate Libraries
Figure 19 Reagent Blue Dye Distribution
24
Material # 20000945
Document # 15049725 v02
Consumables
}
}
}
96-well midi plate
Microseal 'B' adhesive seal
Tris-HCl 10 mM, pH8.5 with 0.1% Tween 20
Preparation
1
If the DNA library plate was stored, thaw it at room temperature, and then centrifuge
at 280 × g for 1 minute.
2
Label wells of a new 96-well midi plate 1–16.
Procedure
1
Transfer 5 µl from each well of the library plate to the corresponding wells of a midi
plate.
2
Normalize each library to 10 nM with Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.
Pipette to mix.
Depending on the quantification yield data of each sample library, the final volume
in the plate can vary from 5–250 µl.
3
Select from the following options:
} For libraries that do not require pooling, the protocol stops here. Proceed to cluster
generation.
} For libraries that require pooling, proceed to Pool Libraries.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
25
Normalize Libraries
Normalize Libraries
Pool Libraries
This process describes how to pool normalized DNA libraries in equal volumes. Do not
perform this process if you are not pooling libraries.
Consumables
}
}
96-well 0.3 ml PCR plate
Microseal 'B' adhesive seal
Procedure
1
Determine the number of samples to combine for each pool. Do not pool samples
with the same index.
2
Transfer 5 µl of each library to be pooled from the library plate to a single well of a
new PCR plate. Pipette to mix.
The total volume in each well is 5 times the number of combined sample libraries.
For example, the volume for 2 samples is 10 µl, the volume for 12 samples is 60 µl,
or the volume for 16 samples is 80 µl.
3
Proceed to cluster generation. For more information, see the system guide for your
Illumina sequencing platform.
SAFE STOPPING POINT
If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.
26
Material # 20000945
Document # 15049725 v02
The protocols described in this guide assume that you have reviewed the contents of this
section, confirmed your kit contents, and obtained all the required consumables and
equipment.
TruSeq Stranded mRNA Library Prep Kit for NeoPrep Kit
Make sure that you have the items identified in this section in your TruSeq Stranded
mRNA Library Prep Kit for NeoPrep (catalog # NP-202-1001) before starting the protocol.
The kit contains 3 boxes.
Box 1, Store at 15°C to 30°C
Quantity
2
1
1
1
1
Description
Library separation tube strips
NeoPrep Oil vial
Oil funnel
Library Card Guide
NeoPrep Library Card, 16-samples
Box 2, Store at -25°C to -15°C
This box contains a TruSeq Stranded mRNA Library Prep for NeoPrep reagent plate
covered by a reagent plate guide.
Reagent Plate Contents
The TruSeq Stranded mRNA Library Prep for NeoPrep reagent plate is a single-use
consumable. It consists of a 96-well foil-sealed plate prefilled with library prep adapters
and reagents for a single TruSeq Stranded mRNA Library Prep for NeoPrep run.
Figure 20 Reagent Plate
Table 3 Default Adapters
Well
Reagent
Description
A
NR006
Adapter Index 6
B
NR013
Adapter Index 13
C
NR012
Adapter Index 12
D
NR014
Adapter Index 14
E
NR005
Adapter Index 5
F
NR015
Adapter Index 15
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
Well
I
J
K
L
M
N
Reagent
NR001
NR010
NR020
NR008
NR025
NR011
Description
Adapter Index 1
Adapter Index 10
Adapter Index 20
Adapter Index 8
Adapter Index 25
Adapter Index 11
27
Supporting Information
Supporting Information
Well
G
H
Reagent
NR019
NR021
Description
Adapter Index 19
Adapter Index 21
Well
O
P
Table 4 Alternate Adapters
Well
Reagent
Description
Q
NR002
Adapter Index 2
R
NR004
Adapter Index 4
S
NR007
Adapter Index 7
T
NR016
Adapter Index 16
Well
U
V
W
X
Reagent
NR018
NR023
Reagent
NR003
NR009
NR022
NR027
Description
Adapter Index 18
Adapter Index 23
Description
Adapter Index 3
Adapter Index 9
Adapter Index 22
Adapter Index 27
NOTE
The large and small reagents and adapters are not the same as manual TruSeq library prep
reagents and adapters. Do not use these reagents for manual TruSeq library prep and do
not use the reagents and adapters in the manual TruSeq library prep kits for NeoPrep
library prep.
Table 5 Large Reagents
Well
Reagent
i
BWS3
ii
ESL
iii
BBS
iv
QDR
v
BWS3
vi
ESL
vii
BBS
viii
–
Table 6
Well
a
b
h
1
2
3
Small Reagents
Reagent Description
FAM
FAM Dye
DBW
Droplet Blue
Water
DBW
Droplet Blue
Water
DBW
Droplet Blue
Water
DBW
Droplet Blue
Water
DBW
Droplet Blue
Water
DBW
Droplet Blue
Water
FAM
FAM Dye
ESL
Elution Solution
LIG4
Ligation Mix
ATL3
A-Tailing Mix
4
ERP4
c
d
e
f
g
28
End Repair Mix
Description
Bead Wash Solution 3
Elution Solution
Bead Binding Solution
Quant Dye Reagent
Bead Wash Solution 3
Elution Solution
Bead Binding Solution
Empty
Well
5
6
Reagent
EPM2
EPM2
Description
Enhanced PCR Mix 2
Enhanced PCR Mix 2
7
PPC2
PCR Primer Cocktail 2
8
ESL
Elution Solution
9
ELB2
Elution Buffer 2
10
BBB2
Bead Binding Buffer 2
11
FSA2
First Strand Synthesis Mix 2
12
13
14
15
BWB2
EPH2
EPH2
SMM2
16
BWB2
Bead Washing Buffer 2
Elute, Prime, Fragment High Mix 2
Elute, Prime, Fragment High Mix 2
Second Strand Marking Master
Mix 2
Bead Washing Buffer 2
Material # 20000945
Document # 15049725 v02
This box is shipped at room temperature. When you receive your kit, store this box at
2°C to 8°C.
Quantity
1
1
1
Reagent
RSB
DMB
RPB2
Description
Resuspension Buffer
Digital Microfluidics Beads
RNA Purification Beads 2
Consumables and Equipment
Make sure that you have the required user-supplied consumables and equipment before
starting the protocol.
The protocol has been optimized and validated using the items listed. Comparable
performance is not guaranteed when using alternate consumables and equipment.
Pipettes and Tips
Use the pipettes and tips specified. Other pipettes and tips are not supported and can
result in reagents not dispensing properly and run failure.
Pipettes and Tips
Supplier
Pipet-Lite XLS+ 8-channel LTS,
2 µl to 200 µl
Rainin, catalog # L8-20XLS+
One of the following 20 µl pipette tips:
• LTS tips 20 µl. Presterilized. Filter
• ART Barrier Pipette Tips 20 µl; 20 µl
SoftFit-L
Pipet-Lite XLS+ 8-channel LTS,
20 µl to 200 µl
One of the following 200 µl pipette tips:
• LTS tips 200 µl. Presterilized. Filter
• ART Barrier Pipette Tips 200 µl; 200 µl
SoftFit-L
• Rainin, catalog # RT-L10F
• Fisher Scientific, catalog # 2749RI
Rainin, catalog # L8-200XLS+
• Rainin, catalog # RT-L200F
• Fisher Scientific, catalog # 2769RI
Consumables
Consumable
Supplier
1000 µl barrier pipette tips
General lab supplier
96-well 0.3 ml skirtless PCR plates, or
Twin.tec 96-well PCR plates
E&K Scientific, part # 480096, or
Eppendorf, part # 951020303
Microseal 'B' adhesive seals
Bio-Rad, part # MSB-1001
RNase/DNase-free multichannel reagent
reservoirs, disposable
VWR, part # 89094-658
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
29
Supporting Information
Box 3, Store at 2°C to 8°C
Consumable
Supplier
[Optional - positive control]
Human UHR total RNA
Agilent Technologies, part # 740000
[Optional - for starting material quality
assessment] One of the following:
• Standard Sensitivity RNA Analysis Kit
(20nt Lower Marker)
• Agilent RNA 6000 Nano Kit
[Optional - for library quality control]
One of the following:
• Standard Sensitivity NGS Fragment
Analysis Kit, 1–6000 bp (500 samples)
• DNA 1000 Kit
[Optional - for library quality control]
One of the following:
• Standard Sensitivity NGS Fragment
Analysis Kit, 1–6000 bp (500 samples)
• DNA 1000 Kit
• Advanced Analytical Technologies,
part # DNF-489
• Agilent Technologies, part # 5067-1511
• Advanced Analytical Technologies,
part # DNF-473-0500
• Agilent Technologies,
part # 5067-1504
• Advanced Analytical Technologies,
part # DNF-473-0500
• Agilent Technologies,
part # 5067-1504
[Optional - for manual normalization]
96-well storage plates, round well,
0.8 ml (midi plate)
Fisher Scientific, part # AB-0859
[Optional - for manual normalization]
Tris-HCl 10 mM, pH 8.5 with 0.1%
Tween 20
General lab supplier
Equipment
30
Equipment
Supplier
96-well thermal cycler (with heated lid)
General lab supplier
NeoPrep Library Prep System
Illumina, catalog # SE-601-1001
Microplate centrifuge
General lab supplier
Vortexer or microplate shaker
General lab supplier
[Optional - for library quality control]
One of the following:
• Fragment Analyzer Automated CE
System
• 2100 Bioanalyzer Desktop System
• Advanced Analytical Technologies,
part # FSv2-CE2 or FSv2-CE10
• Agilent Technologies, part # G2940CA
Material # 20000945
Document # 15049725 v02
The TruSeq Stranded mRNA Library Prep Kit for NeoPrep contains the following index
adapter sequences.
} The indexes are TruSeq LT single-index adapters.
} The index numbering is not contiguous. There is no Index 17, 24, or 26.
} The sequence contains 7 bases. The seventh base, shown in parenthesis (), is not
included in the Index Read. Record only the first 6 bases in a sample sheet. For
indexes 13 and above, the seventh base (in parentheses) might not be A, which is
seen in the cycle 7 of the Index Read.
For more information on the number of cycles used to sequence the Index Read, see
the system guide for your Illumina sequencing platform.
Table 7 Indexed Adapter Sequences
Adapter
Sequence
Adapter
Sequence
NR0001
ATCACG(A)
NR0013
AGTCAA(C)
NR0002
CGATGT(A)
NR0014
AGTTCC(G)
NR0003
TTAGGC(A)
NR0015
ATGTCA(G)
NR0004
TGACCA(A)
NR0016
CCGTCC(C)
NR0005
ACAGTG(A)
NR0018
GTCCGC(A)
NR0006
GCCAAT(A)
NR0019
GTGAAA(C)
NR0007
CAGATC(A)
NR0020
GTGGCC(T)
NR0008
ACTTGA(A)
NR0021
GTTTCG(G)
NR0009
GATCAG(A)
NR0022
CGTACG(T)
NR0010
TAGCTT(A)
NR0023
GAGTGG(A)
NR0011
GGCTAC(A)
NR0025
ACTGAT(A)
NR0012
CTTGTA(A)
NR0027
ATTCCT(T)
Acronyms
Acronym
Definition
ATL
A-Tailing Mix
BBB
Bead Binding Buffer
BBS
Bead Binding Solution
BWB
Bead Washing Buffer
BWS
Bead Wash Solution
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
31
Supporting Information
Index Adapter Sequences
32
Acronym
Definition
DBW
Droplet Blue Water
DMB
Digital Microfluidics Beads
ELB
Elution Buffer
EPH
Elute, Prime, Fragment High Mix
EPM
Enhanced PCR Mix
ERP
End Repair Mix
ESL
Elution Solution
FAM
FAM Dye
FSA
First Strand Synthesis Mix
LIG
Ligation Mix
PPC
PCR Primer Cocktail
QDR
Quant Dye Reagent
RSB
Resuspension Buffer
SMM
Second Strand Marking Master Mix
Material # 20000945
Document # 15049725 v02
Document
Date
Document # 15049725
v02
Material # 20000945
March
2016
Document # 15049725
v01
January
2016
Part # 15049725 Rev. C
June
2015
Part # 15049725 Rev. B
April
2015
Part # 15049725 Rev. A
March
2015
Description of Change
• Corrected order of steps in Load the Library Card section to
pipette to mix samples in the sample plate before samples are
loaded into the library card.
• Removed quantification and normalization procedures and
information
• Added Custom Protocol Selector to Additional Resources
• Moved Revision History to back of booklet
• Clarify to mix by pipette when diluting RNA in Prepare Samples for
Loading
• Changed loading order to load samples first
• Changed loading workflow on reagent plate guide and library
card guide
• Added 3 minute wait to drain oil from vial into library card
• Removed step to pipette samples before transferring to library
card
• Renamed button Home at the conclusion of unloading libraries
• Consumables table:
• Removed 20 µl and 200 µl pipettes. They are specified in
Pipettes and Tips.
• Removed 1000 µl pipettes as they are standard lab items
• Added kits for starting material quality assessment
• Moved Acronyms to end of Supporting Information
Added required pipettes to:
• Pipette Tip Requirements
• Consumables and Equipment
Removed part numbers and package layouts from Kit Contents
Updated reagent names in Reagent Plate Contents
Added required pipette tip and calibration information to:
• New Pipette Tip Requirements
• Consumables and Equipment
• Tips and Techniques
• Load the Library Card
Changed BaseSpace resource reference to helpcenter
Initial release.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
33
Revision History
Revision History
Notes
For technical assistance, contact Illumina Technical Support.
Table 8 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 9 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Japan
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
China
400.635.9898
Singapore
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
Taiwan
Hong Kong
800960230
United Kingdom
Ireland
1.800.812949
Other countries
Italy
800.874909
Contact Number
0800.111.5011
0800.0223859
0800.451.650
800.16836
1.800.579.2745
900.812168
020790181
0800.563118
00806651752
0800.917.0041
+44.1799.534000
Safety data sheets (SDSs)—Available on the Illumina website at
support.illumina.com/sds.html.
Product documentation—Available for download in PDF from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
TruSeq Stranded mRNA Library Prep for NeoPrep Reference Guide
Technical Assistance
Technical Assistance
*20000945*
Material # 20000945
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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