TruSeq Stranded mRNA Library Prep
for NeoPrep
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
Prepare Samples for Loading
Set Up Run and Load Library Card
Unload Libraries
Validate Libraries
Normalize Libraries
Pool Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Document # 15059581 v02
March 2016
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Preparation
1
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
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 Protocol Guide
3
Prepare Samples for Loading
Prepare Samples for Loading
Set Up Run and Load Library Card
Procedure
1
Vortex the reagent plate for 3 seconds.
2
Centrifuge at 600 × g for 5 seconds.
3
Select Prepare Libraries on the Library Prep Instrument 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.
6
Review the run and sample information. Select Next.
7
Enter the consumable tracking information. Select Next.
8
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.
10 Place the library card guide on the library card.
11 Load the entire contents of the oil vial into the library card using the oil funnel.
WARNING
Use the required pipette tips. 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.
12 Insert pipette tips to the bottom of the wells of the prepared sample plate. Pipette up
and down 1 time to mix.
13 Transfer 25 µl of prepared samples 1–8.
14 Transfer 25 µl of prepared samples 9–16.
15 If you are preparing < 16 samples, add 25 µl RSB to empty sample wells.
16 Transfer 125 µl of the large reagents i–iv.
17 Transfer 125 µl of the large reagents v–vii.
18 Vortex DMB until well-dispersed.
19 Add 80 µl DMB to the large reagent well viii.
20 Transfer 15 µl of small reagents 1–4, and then 5–8.
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Document # 15059581 v02
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.
22 For small reagents 13–16:
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.
23 Transfer 5 µl of small reagents a–d, and then e–h.
24 Transfer 3 µl of adapters A–H.
25 Transfer 3 µl of adapters I–P.
26 Remove the library card guide.
WARNING
To avoid instrument damage, make sure that the library card guide is removed from
the library card.
27 Close the library card compartment door. Select Start Run.
28 When the run is complete, select Next.
TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide
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Set Up Run and Load Library Card
21 For small reagents 9–12:
Unload Libraries
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.
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.
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.
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.
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Document # 15059581 v02
Procedure
1
Quantify the libraries using qPCR according to the Illumina Sequencing Library qPCR
Quantification Guide (document # 11322363).
2
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.
3
If using a DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer, run 1 µl
undiluted DNA library.
4
Check the size and purity of the sample. Expect the final product to be a band at
~300 bp.
TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide
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Validate Libraries
Validate Libraries
Normalize Libraries
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.
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.
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Document # 15059581 v02
Procedure
1
Determine the number of samples to combine for each pool.
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.
3
Proceed to cluster generation.
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 Protocol Guide
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Pool Libraries
Pool Libraries
Acronyms
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Acronym
Definition
DMB
Digital Microfluidics Beads
RPB2
RNA Purification Beads 2
RSB
Resuspension Buffer
Document # 15059581 v02
For technical assistance, contact Illumina Technical Support.
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TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide
Technical Assistance
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