Thermo Fisher Scientific Oncomine Immune Response Research Assay User guide

Brand
Thermo Fisher Scientific
Model
Oncomine Immune Response Research Assay
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
Oncomine™ Immune Response Research
Assay
USER GUIDE
Catalog Number A32881
Publication Number MAN0015867
Revision B.0
Manufacturer: Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated with
the workflow covered in this guide.
Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,
INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR
USE OF IT.
Revision history: Pub. No. MAN0015867
Revision Date Description
B.0 31 May 2017 Include supplementary information for performing functional RNA
quantification. Correction to the amount of 2X Oncomine™ Immune
Response Research Panel– Chef Ready that is provided.
A.0 31 October 2016
Oncomine
™
Immune Response Research Assay User Guide
. Provides
instruction for library preparation, templating, sequencing, and data
analysis of Oncomine™ Immune Response Research Assay libraries.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2017 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 Product information ....................................... 6
Product description ............................................................. 6
Contents and storage ............................................................ 7
Oncomine™ Immune Response Research Assay ................................. 7
Oncomine™ Immune Response Research Assay– Chef Ready ..................... 8
Required materials not supplied .................................................. 9
Recommended materials ....................................................... 10
■CHAPTER 2 Before you begin .......................................... 11
Procedural guidelines .......................................................... 11
Before each use of the kit ....................................................... 11
Workflow overview ............................................................. 12
■CHAPTER 3 Library preparation ....................................... 13
Guidelines for RNA isolation and quantification .................................... 13
Reverse transcription and target amplification ..................................... 13
Reverse transcribe the RNA ................................................. 13
(Chef-ready kit only)
Transfer cDNA to an IonCode™ 96‑well plate ................ 14
Prepare cDNA target amplification reactions .................................. 15
Amplify the targets ........................................................ 16
Partially digest amplicons .................................................. 16
Ligate adapters to the amplicons and purify ....................................... 17
Ion Xpress™ adapters only: Combine and dilute adapters ....................... 17
Perform ligation reaction ................................................... 18
Purify the library .......................................................... 18
Quantify the library by qPCR ..................................................... 20
Elute the library ........................................................... 20
Quantify the library by qPCR and calculate the dilution factor .................... 20
Store libraries ............................................................. 22
Quantify the amplified library with the Qubit™ Fluorometer .......................... 23
Amplify the library ......................................................... 23
Purify the amplified library ................................................. 23
Qubit™ Fluorometer: Quantify the library and calculate the dilution factor ......... 25
Store libraries ............................................................. 26
Oncomine
™
Immune Response Research Assay User Guide
3
Combine libraries .............................................................. 26
Guidelines for templating and sequencing ......................................... 27
■CHAPTER 4 Create an assay-specific Planned Run ................. 28
About planned runs ............................................................ 28
Create a custom Planned Run template ........................................... 29
Create a planned run ........................................................... 30
■CHAPTER 5 Data analysis .............................................. 32
Data analysis overview .......................................................... 32
Gene expression level quantification with the immuneResponseRNA plugin ........... 32
Review run results ............................................................. 33
Sample Characteristics tab ................................................. 34
Sample Correlation tab ..................................................... 37
Analysis of Selected Targets ................................................. 38
Analysis of All Targets tab .................................................. 39
Downloadable reports ...................................................... 40
Affymetrix™ Transcriptome Analysis Console (TAC) analysis ......................... 41
System requirements ...................................................... 41
Download and install TAC software ........................................... 41
Perform TAC expression analysis ............................................ 42
■APPENDIX A Tips and troubleshooting ............................... 45
Tips .......................................................................... 45
Troubleshooting ............................................................... 46
Library yield and quantification .............................................. 46
Other ..................................................................... 47
■APPENDIX B Supplemental information .............................. 49
Functional RNA quantification ................................................... 49
Install preloaded Reference Library file ........................................... 52
Install Target Regions .bed files .................................................. 52
Manually launch the immuneResponseRNA plugin ................................. 53
■APPENDIX C Safety ..................................................... 55
Chemical safety ................................................................ 56
Biological hazard safety ......................................................... 57
Contents
4
Oncomine
™
Immune Response Research Assay User Guide
■Documentation and support ............................................. 58
Obtain information from the Help system ......................................... 58
Customer and technical support ................................................. 58
Limited product warranty ....................................................... 58
Contents
Oncomine
™
Immune Response Research Assay User Guide
5
Product information
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
The Oncomine™ Immune Response Research Assay is an RNA‑based multi‑biomarker
next‑generation sequencing (NGS) assay that enables the characterization of gene
expression levels from approximately 400 genes relevant to oncology and immune
therapy response research.
This guide covers the following products:
• Oncomine™ Immune Response Research Assay (Cat. No. A32881)
• Ion AmpliSeq™ Library Kit 2.0 (Cat. No. 4475345)
• SuperScript™ VILO™ cDNA Synthesis Kit (Cat. No. 11754050)
• IonCode™ Barcode Adapters (Cat. No. A29751)
• Ion Xpress™ Barcode Adapters (various Cat. Nos.)
• Ion AmpliSeq™ Sample ID Panel (Cat. No. 4479790)
SuperScript™ and Ion AmpliSeq™ products include reagents to prepare targeted
libraries from RNA for sequencing on the Ion S5™/Ion S5™ XL or Ion PGM™ Systems.
The kits are designed for use with barcoded adapters so that multiple libraries can be
combined and loaded onto a single Ion Chip to minimize the per‑sample sequencing
cost.
Note: The Oncomine™ Immune Response Research Assay– Chef Ready library
preparation kit (Cat. No. A32928) is also available for automated library preparation
(see “Oncomine™ Immune Response Research Assay– Chef Ready“ on page 8). The
kit provides Oncomine™ Immune Response Research Panel– Chef Ready at 2X
concentration pre‑measured in barcoded Primer Pool tubes ready to load into an Ion
AmpliSeq™ Chef Reagents DL8 cartridge.
1
6
Oncomine
™
Immune Response Research Assay User Guide
Contents and storage
The Oncomine™ Immune Response Research Assay (Cat. No. A32881) consists of the
Oncomine™ Immune Response Research Panel and the Ion AmpliSeq™ Library Kit 2.0
for the rapid preparation of barcoded sample libraries from RNA, which you can then
combine and sequence simultaneously.
Contents Amount Storage
Oncomine™ Immune Response Research Assay (Part No. A31930)
5X Oncomine™ Immune Response Research
Panel
4 × 32 µL −25°C to −15°C
Ion AmpliSeq™ Library Kit 2.0 (Part No. 4475345)
5X Ion AmpliSeq™ HiFi Mix (red cap) 32 µL –30ºC to –10ºC
FuPa Reagent (brown cap) 16 µL
Switch Solution (yellow cap) 32 µL
DNA Ligase (blue cap) 16 µL
Ion AmpliSeq™ Adapters (green cap) 16 µL
Platinum™ PCR SuperMix HiFi (black cap) 400 µL
Library Amplification Primer Mix (white cap) 16 µL
Low TE (clear cap) 1.7 mL 15°C to 30°C[1]
[1] Can be stored at –30ºC to –10ºC for convenience.
Oncomine™
Immune Response
Research Assay
Chapter 1 Product information
Contents and storage
1
Oncomine
™
Immune Response Research Assay User Guide
7
The Oncomine™ Immune Response Research Assay– Chef Ready Kit (Cat. No. A32928,
ordered separately) provides Oncomine™ Immune Response Research Panel– Chef
Ready at 2X concentration pre‑measured in barcoded Primer Pool tubes ready to load
into an Ion AmpliSeq™ Chef Reagents DL8 cartridge. In addition, the kit provides all
the reagents and supplies in an Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024)
sucient for preparing 32 libraries. See the Ion AmpliSeq™ Library Preparation on the Ion
Chef™ System User Guide (Pub. No. MAN0013432) for detailed information on
preparing Oncomine™ Immune Response Research Assay libraries on the Ion Chef™
System.
Component Quantity per kit Storage
Oncomine™ Immune Response Research Panel– Chef Ready (Part. No. A32929)
2X Oncomine™ Immune Response Research Panel–
Chef Ready
8 × 150 µL –30°C to –10°C
Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024)
Ion AmpliSeq™ Chef Reagents DL8 (Part No. A29025) 4 cartridges –30°C to –10°C
Ion AmpliSeq™ Chef Solutions DL8 (Part No. A29026) 4 cartridges 2°C to 8°C[1]
Ion AmpliSeq™ Chef Supplies DL8 (per insert)
(Part No. A29027)
• Ion AmpliSeq™ Tip Cartridge L8
• PCR Frame Seal
• Enrichment Cartridge
1 box with
4 inserts
15°C to 30°C
IonCode™ 0101–0132 in 96 Well PCR Plates (dried)
(Part No. A29028)
Set includes 4 PCR plates:
• IonCode™ 0101–0108 in 96 Well PCR Plate (red)
• IonCode™ 0109–0116 in 96 Well PCR Plate
(yellow)
• IonCode™ 0117–0124 in 96 Well PCR Plate
(green)
• IonCode™ 0125–0132 in 96 Well PCR Plate (blue)
1 set of 4 plates 15°C to 30°C
[1] Ion AmpliSeq™ Chef Solutions DL8 cartridges are shipped at ambient temperature, but need to be stored at
2°C to 8°C upon arrival.
Oncomine™
Immune Response
Research Assay–
Chef Ready
Chapter 1 Product information
Contents and storage
1
8
Oncomine
™
Immune Response Research Assay User Guide
Required materials not supplied
Unless otherwise indicated, all materials are available through thermosher.com.
MLS: Fisher Scientic (sherscientic.com) or other major laboratory supplier.
Item Source
SuperScript™ VILO™ cDNA Synthesis Kit 11754050
IonCode™ Barcode Adapters 1–384 Kit
OR
Ion Xpress™ Barcode Adapters
A29751
4471250
Agencourt™ AMPure™ XP Kit Beckman Coulter, A63880 or
A63881
One of the following:
• GeneAmp™ PCR System 9700 or Dual 96-well Thermal
Cycler
• AB™ 2720 Thermal Cycler
• Veriti™ 96-well Thermal Cycler
• ProFlex™ 96‑well PCR System
See web product pages
MicroAmp™ Optical 96-well Reaction Plate N8010560
4306737 (with barcode)
MicroAmp™ Fast Optical 96-Well Reaction Plate 4346907
MicroAmp™ Optical Adhesive Film 4311971
MicroAmp™ Adhesive Film 4306311
MicroAmp™ Compression Pad 4312639
DynaMag™-96 Side Magnet, or other plate magnet 12331D
Non-stick RNase-Free Microfuge Tubes MLS
Low TE buffer MLS
Nuclease-free Water AM9932
Absolute ethanol MLS
Pipettors, 2–200 μL, and low-retention filtered pipette tips MLS
Chapter 1 Product information
Required materials not supplied
1
Oncomine
™
Immune Response Research Assay User Guide
9
Recommended materials
Unless otherwise indicated, all materials are available through thermosher.com.
MLS: Fisher Scientic (sherscientic.com) or other major laboratory supplier.
Item Source
Recommended additional equipment
Real-time PCR instrument (e.g., Applied Biosystems™
7900HT, 7500, StepOnePlus™, ViiA™ 7 Systems, or a
QuantStudio™ Real-Time PCR System)
See web product pages
96-well plate centrifuge MLS
Recommended for nucleic acid isolation
RecoverAll™ Multi-Sample RNA/DNA Workflow A26069
RecoverAll™ Total Nucleic Acid Isolation Kit AM1975
Recommended for library quantification
Ion Library TaqMan® Quantitation Kit 4468802
Qubit™ 3.0 Fluorometer or Qubit™ 2.0 Fluorometer[1] and
Qubit™ dsDNA HS Assay Kit (DNA)
Q33216, Q32851/Q32854
Recommended for functional RNA quantification
Promyelocytic Leukemia (HL-60) Total RNA AM7836
TaqMan® Fast Virus 1-Step Master Mix 4444432
TaqMan® Gene Expression Assay, GUSB (20X, Hs00939627) 4331182
[1] Supported but no longer available for purchase.
Chapter 1 Product information
Recommended materials
1
10
Oncomine
™
Immune Response Research Assay User Guide
Before you begin
Procedural guidelines
• Minimize freeze‑thaw cycles of Oncomine™ Immune Response Research Assay
panels by aliquoting as needed for your experiments. Panels can be stored at 4°C
for one year.
• Use good laboratory practices to minimize cross‑contamination of products. If
possible, perform PCR setup in an area or room that is free of amplicon
contamination. Always change pipee tips between samples.
•Use a calibrated thermal cycler specied in “Required materials not supplied“.
• Pipet viscous solutions, such as 5X Ion AmpliSeq™ HiFi Mix, FuPa Reagent,
Switch Solution, DNA Ligase, and panels, slowly and ensure complete mixing by
vortexing or pipeing up and down several times.
Before each use of the kit
• Thaw components that contain enzymes—such as 5X Ion AmpliSeq™ HiFi Mix,
FuPa Reagent, DNA Ligase, and Platinum™ PCR SuperMix HiFi—on ice, and
keep on ice during procedure. All other components, including primer pools, can
be thawed at room temperature. Gently vortex and centrifuge before use.
• If there is visible precipitate in the Switch Solution after thawing, vortex or pipet
up and down at room temperature to resuspend.
2
Oncomine
™
Immune Response Research Assay User Guide
11
Workflow overview
RNA
cDNA
P1
P1
X
Barcode Adapters
Barcoded library
X
Primer pairs
Amplicons
Reverse transcribe
Amplify targets
Partially digest amplicons
Ligate adapters
Quantify libraries
Combine libraries (optional)
Isolate and quantify RNA
Chapter 2 Before you begin
Workflow overview
2
12
Oncomine
™
Immune Response Research Assay User Guide
Library preparation
IMPORTANT! We recommend Oncomine™ Immune Response Research Assay – Chef
Ready Kit (Cat. No. A32928) users perform the reverse transcription as described in
the following section “Reverse transcribe the RNA“. Following completion of cDNA
synthesis refer to the Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User
Guide (Pub. No. MAN0013432) for instructions to prepare Oncomine™ Immune
Response Research Assay libraries on the Ion Chef™ System.
Guidelines for RNA isolation and quantification
• Use kits in “Recommended materials“ on page 10 for isolating total RNA.
•We recommend verifying sample RNA integrity by performing the functional
RNA quantication, see page 49 for more information.
• We recommend the Qubit™ RNA HS Assay Kit (Cat. No. Q32855) for quantifying
RNA.
• Each reverse transcription reaction requires 10 ng of DNase‑treated total RNA
(≥1.43 ng/μL).
• In general, library yield from high quality RNA is greater than from degraded
samples. Library yield is not indicative of sequencing performance.
• Increasing the amount of RNA will usually result in higher quality libraries,
especially when RNA quality or quantity is unknown.
Reverse transcription and target amplification
1. If the RNA was prepared from FFPE tissue and not previously heat‑treated, heat
at 80°C for 10 minutes, then cool to room temperature.
2. If there is visible precipitate in the 5X VILO™ Reaction Mix tube or cap after
thawing, vortex or pipet up and down at room temperature to resuspend before
pipeing.
3
Reverse
transcribe the
RNA
Oncomine
™
Immune Response Research Assay User Guide
13
3. For each sample, add the following components into a single well of a 96‑well
PCR plate. Prepare a master mix without sample RNA for multiple reactions.
Component Volume
5X VILO™ Reaction Mix 2 µL
10X SuperScript™ Enzyme Mix 1 µL
Total RNA (10 ng)[1] ≤ 7 µL
Nuclease-free water to 10 µL
Total volume per well 10 µL
[1] Substitute an equal volume of nuclease-free water or low TE to prepare a non-template control (NTC).
RNA plate
1 2 34 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Sample
NTC
4. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, then briey
centrifuge to collect droplets. Alternatively, mix by pipeing at least half the total
volume up and down at least ve times before sealing the plate.
5. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal
cycler, then run the following program to synthesize cDNA.
Temperature Time
25°C 10 minutes
42°C 60 minutes
85°C 5 minutes
4°C Hold
STOPPING POINT Samples can be stored at 4°C for up to 16 hours in the thermal
cycler. For longer term, store at −20°C.
6. Centrifuge the plate briey to collect any droplets at the boom of the wells, then
proceed to the next step.
Oncomine™ Immune Response Research Assay (Cat. No. A32881) users performing
manual library preparation proceed to “Prepare cDNA target amplication
reactions“, without transferring the cDNA synthesis reactions.
1. Place the 96‑well plate in a pre‑chilled cold block.
2. Remove the seal from the plate, then transfer each individual cDNA synthesis
reaction to the corresponding row in column 1 of an IonCode™ 96‑well plate.
(Chef-ready kit
only)
Transfer
cDNA to an
IonCode™ 96‑well
plate
Chapter 3 Library preparation
Reverse transcription and target amplification
3
14
Oncomine
™
Immune Response Research Assay User Guide
3. Pipet 5 μL nuclease‑free water into each transferred cDNA synthesis reaction in
column 1 of the IonCode™ 96‑well plate.
1 2 34 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
1 2 34 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
RNA plate
12
IonCode plate
1Each column 1 well contains 10-µL cDNA synthesis reaction, and 5‑µL nuclease-free
water.
2Each column 6 well contains a dried-down IonCode™ barcode. Lowest barcode
number is in A6 and the highest is in H6. All appear light blue in the actual plate.
4. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, then
briey centrifuge to collect droplets. Alternatively, mix by pipeing at least half
the total volume up and down at least 5 times before sealing the plate.
Proceed to "Thaw the reagents and prepare the instrument" in the Ion AmpliSeq™
Library Preparation on the Ion Chef™ System User Guide (Pub. No. MAN0013432).
Information required to set up the Ion Chef™ Instrument run is in the following table.
# of primer pools Target amplification cycles Anneal & extension time
1 20 4 minutes
IMPORTANT! Primer pools and HiFi Mix are viscous. Pipet slowly and mix
thoroughly.
1. Place the 96‑well plate in a pre‑chilled cold block or on ice.
2. Thaw the 5X Ion AmpliSeq™ HiFi Mix on ice, gently vortex to mix, then
centrifuge briey to collect.
3. Remove the seal from the plate, then add the following components to each
cDNA synthesis reaction. Prepare a master mix for multiple reactions.
Component Volume
5X Ion AmpliSeq™ HiFi Mix (red cap) 4 µL
5X Oncomine™ Immune Response Research Panel 4 µL
Nuclease-free Water 2 µL
Total volume per well (includes 10 µL from cDNA synthesis) ~20 µL
4. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, then
briey centrifuge to collect droplets. Alternatively, mix by pipeing at least half
the total volume up and down at least 5 times before sealing the plate.
Prepare cDNA
target
amplification
reactions
Chapter 3 Library preparation
Reverse transcription and target amplification
3
Oncomine
™
Immune Response Research Assay User Guide
15
Proceed to “Amplify the targets“.
1. Place a MicroAmp™ Compression Pad on the plate, then load the plate into the
thermal cycler.
2. Run the following program to amplify the target regions.
Stage Step Temperature Time
Hold Activate the
enzyme
99°C 2 min
19 cycles Denature 99°C 15 sec
Anneal and extend 60°C 4 min
Hold — 10°C Hold
Note: Addition of the Ion AmpliSeq™ Sample ID Panel does not require
adjustment to cycle number.
IMPORTANT! When amplifying multiple samples in a single PCR plate, make
sure that the input across all samples is roughly equivalent so that the selected
cycle number is optimal for all the samples in the run.
3. Remove the plate from the thermal cycler, then briey centrifuge the plate to
collect the contents.
STOPPING POINT Target amplication reactions may be stored at 10°C overnight
on the thermal cycler. For longer periods, store at −20°C.
IMPORTANT! Keep the plate on ice or in a pre‑chilled 4°C cold block while preparing
the reactions.
1. Keep the FuPa Reagent (brown cap) on ice, gently vortex to mix, then briey
centrifuge to collect.
2. Carefully remove the adhesive lm from the plate.
IMPORTANT! Be careful when removing the lm to minimize contamination.
3. Add 2 μL of FuPa Reagent to each amplied sample. The total volume is ~22 μL.
4. Seal the plate with a new clear adhesive lm, vortex thoroughly, then centrifuge
briey to collect droplets. Alternatively, mix by pipeing at least half the total
volume up and down at least 5 times before sealing the plate.
Amplify the
targets
Partially digest
amplicons
Chapter 3 Library preparation
Reverse transcription and target amplification
3
16
Oncomine
™
Immune Response Research Assay User Guide
5. Place a compression pad on the plate, load in the thermal cycler, then run the
following program:
Temperature Time
50°C 10 min
55°C 10 min
60°C 20 min
10°C Hold (for up to 1 hour)
6. Briey centrifuge the plate to collect the contents to the boom of the wells.
Ligate adapters to the amplicons and purify
When sequencing multiple libraries on a single chip, you must ligate a dierent
barcode adapter to each library. DNA and RNA libraries from the same sample also
require dierent barcodes.
IonCode™ Adapters are provided at the appropriate concentration and include
forward and reverse adapters in a single well. No further handling is necessary.
Ion Xpress™ adapters require handling and dilution as described below.
IMPORTANT! When handling barcoded adapters, be careful to avoid cross
contamination by changing gloves frequently and opening one tube at a time.
For each barcode X selected, prepare a mix of Ion P1 Adapter and Ion Xpress™
Barcode X at a nal dilution of 1:4 for each adapter. For example, combine the
volumes indicated in the following table. Scale volumes as necessary. Use 2 μL of this
barcode adapter mix in step 3 below.
Component Volume
Ion P1 Adapter 2 µL
Ion Xpress™ Barcode X[1] 2 µL
Nuclease-free Water 4 µL
Total 8 µL
[1] X = barcode chosen
Note: Store diluted adapters at –20°C.
Ion Xpress™
adapters only:
Combine and
dilute adapters
Chapter 3 Library preparation
Ligate adapters to the amplicons and purify
3
Oncomine
™
Immune Response Research Assay User Guide
17
1. If there is visible precipitate in the Switch Solution or the tube cap after thawing,
vortex or pipet up and down at room temperature to resuspend before pipeting.
2. Carefully remove the plate seal, then add the following components in the order
listed to each well containing digested amplicons.
IMPORTANT! Add the DNA Ligase last. Do not combine DNA Ligase and
adapters before adding to digested amplicons.
Order of
addition Component Volume
1 Switch Solution (yellow cap) 4 µL
2 Ion Xpress™ barcode adapter mix 2 µL
—Total volume (including ~22 µL of digested amplicon) ~28 µL
3. Seal the plate with a new MicroAmp™ Adhesive Film, vortex thoroughly, then
briey centrifuge to collect droplets.
4. Carefully remove the plate seal, then add 2 μL of DNA Ligase (blue cap) to each
well.
5. Seal the plate with a new lm, vortex thoroughly, then briey centrifuge to
collect droplets.
6. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal
cycler, then run the following program:
Temperature Time
22°C 30 minutes
72°C 10 minutes
10°C Hold (for up to 60 minutes)
STOPPING POINT Samples can be stored for longer periods at –20°C.
IMPORTANT! Bring Agencourt™ AMPure™ XP Reagent to room temperature and
vortex thoroughly to disperse the beads before use. Pipet the solution slowly.
Do NOT substitute a Dynabeads™‑based purication reagent for the Agencourt™
AMPure™ XP Reagent.
1. Prepare 70% ethanol (350 μL × # of samples) fresh daily.
2. Briey centrifuge the plate to collect the contents in the boom of the wells.
3. Carefully remove the plate seal, then add 45 μL (1.5X sample volume) of
Agencourt™ AMPure™ XP Reagent to each library. Pipet up and down 5 times to
mix the bead suspension with the DNA thoroughly.
Note: Visually inspect each well to ensure that the mixture is homogeneous.
4. Incubate the mixture for 5 minutes at room temperature.
Perform ligation
reaction
Purify the library
Chapter 3 Library preparation
Ligate adapters to the amplicons and purify
3
18
Oncomine
™
Immune Response Research Assay User Guide
5. Place the plate in a magnetic rack such as the DynaMag™‑96 Side Magnet, then
incubate for 2 minutes or until the solution clears. Carefully remove, then discard
the supernatant without disturbing the pellet.
6. Add 150 μL of freshly prepared 70% ethanol, then move the plate side‑to‑side in
the two positions of the magnet to wash the beads. Carefully remove, then
discard the supernatant without disturbing the pellet.
Note: If your magnet does not have two positions for shifting the beads, remove
the plate from the magnet and gently pipet up and down 5 times (with the
pipeor set at 100 μL), then return the plate to the magnet and incubate for
2 minutes or until the solution clears.
7. Repeat step 6 for a second wash.
8. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in
the magnet, air‑dry the beads at room temperature for 2−5 minutes. Do not
overdry.
IMPORTANT! Residual ethanol drops inhibit library amplication. If needed,
centrifuge the plate and remove remaining ethanol before air‑drying the beads.
Under conditions of low relative humidity the beads air‑dry rapidly. Do not
overdry.
Proceed immediately to “Quantify the library by qPCR“ on page 20
Note: Alternatively, quantify the libraries using a Qubit™ 2.0 or 3.0 Fluorometer, see
“Quantify the amplied library with the Qubit™ Fluorometer“ on page 23.
Chapter 3 Library preparation
Ligate adapters to the amplicons and purify
3
Oncomine
™
Immune Response Research Assay User Guide
19
Quantify the library by qPCR
Elute the library, then determine the concentration by qPCR with the Ion Library
TaqMan® Quantitation Kit (Cat. No. 4468802). Libraries that have not undergone a
second round of amplication typically have yields of 100–500 pM. However, yield is
not indicative of library quality. After quantication, determine the dilution factor that
results in a concentration of ~50 pM.
1. Remove the plate with puried libraries from the plate magnet, then add 50 μL
of Low TE to the pellet to disperse the beads.
2. Seal the plate with MicroAmp™ Adhesive Film, vortex thoroughly, then briey
centrifuge to collect droplets. Alternatively, mix by pipeing at least half the total
volume up and down at least 5 times before sealing the plate.
3. Incubate at room temperature for at least 2 minutes.
4. Place the plate on the magnet for at least 2 minutes.
STOPPING POINT Libraries can be stored at 4–8°C for up to 1 month. For longer
term, store at –20°C. We recommend transferring the supernatant to a 1.5‑mL
Eppendorf LoBind™ tube for long‑term storage.
5. Prepare a 100‑fold dilution for quantication. Remove 2 μL of supernatant,
containing the library, then combine with 198 μL of Nuclease‑free Water.
Determine the concentration of each Oncomine™ Immune Response Research Assay
library by qPCR with the Ion Library TaqMan® Quantitation Kit using the following
steps. Analyze each sample, standard, and negative control in duplicate reactions.
1. Prepare three 10‑fold serial dilutions of the E. coli DH10B Ion Control Library
(~68 pM; from the Ion Library TaqMan® Quantitation Kit) at 6.8 pM, 0.68 pM, and
0.068 pM. Mark these tubes as standards, then use these concentrations in the
qPCR instrument software.
2. Calculate, then prepare the required volume of PCR master mix for duplicate
reactions of each library sample, standard, and NTC using the following table.
Include a 5−10% overage to accommodate pipeing errors.
Component Volume per reaction
96-well plate 384-well plate
2X TaqMan® Master Mix 10 µL 5 µL
20X Ion TaqMan® Assay 1 µL 0.5 µL
Total 11 µL 5.5 µL
Elute the library
Quantify the
library by qPCR
and calculate the
dilution factor
Chapter 3 Library preparation
Quantify the library by qPCR
3
20
Oncomine
™
Immune Response Research Assay User Guide
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Thermo Fisher Scientific Oncomine Immune Response Research Assay User guide

Brand
Thermo Fisher Scientific
Model
Oncomine Immune Response Research Assay
Type
User guide
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