PN 100-7135 B1 Protocol - University of Sydneybosch.test.med.usyd.edu.au/facilities/molecular... ·...
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Fluidigm Corporation 1 f Protocol Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Required Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Suggested Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Suggested Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Suggested Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 IFC Types and Related Scripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Best Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Preparation for Cell Capture and Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Preparation for DNA Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Use the Chip Map Loading Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Prime the IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Prepare Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Prepare Reagent Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Run Lysis and DNA Amplification on the C 1 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 (Optional) Prepare the Tube Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Harvest the Amplified DNA Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Quantify Harvest Amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Targeted Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Appendix 1: IFC (Chip) Pipetting Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Appendix 2: C 1 Single-Cell Auto Prep Reagent Kit for DNA Seq (PN 100-7357) . . . . . . . . . . . . . . 43 Appendix 3: How to Use Barrier Tape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Using the C 1 ™ Single-Cell Auto Prep System to Generate Single-Cell Libraries for DNA Sequencing PN 100-7135 B1
Transcript of PN 100-7135 B1 Protocol - University of Sydneybosch.test.med.usyd.edu.au/facilities/molecular... ·...
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f
ProtocolIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Required Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Suggested Consumables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Suggested Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Suggested Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10IFC Types and Related Scripts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Best Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Preparation for Cell Capture and Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Preparation for DNA Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Use the Chip Map Loading Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Prime the IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Prepare Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Prepare Reagent Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Run Lysis and DNA Amplification on the C1 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25(Optional) Prepare the Tube Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Harvest the Amplified DNA Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Quantify Harvest Amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Targeted Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Appendix 1: IFC (Chip) Pipetting Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Appendix 2: C1 Single-Cell Auto Prep Reagent Kit for DNA Seq (PN 100-7357) . . . . . . . . . . . . . . 43Appendix 3: How to Use Barrier Tape. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Using the C1™ Single-Cell Auto Prep System to Generate
Single-Cell Libraries for DNA Sequencing
PN 100-7135 B1
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Introduction
This protocol describes how you perform DNA sequencing from single cells. First, whole genome amplification (WGA) is performed with the C1 Single-Cell Auto Prep System (“C1
™ System”) and the C1™
Single-Cell Auto Prep Integrated Fluidic Circuit (IFC).
Whole genome amplification as described in this protocol is achieved through Multiple Displacement Amplification with the illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Life Sciences). After cells are lysed and the DNA denatured, random hexamers anneal to single stranded DNA. Phi29 polymerase is used to amplify the randomly primed regions of the genome. The amplification process continues with the displacement of the DNA strand and annealing of random hexamers, generating a high single-cell genomic yield for analysis. Multiple Displacement Amplification is isothermal and has a low error rate due to the proof-reading capability of the Phi29 polymerase. The average product length with Multiple Displacement Amplification is ~10kb, and the average yield using a C1™ IFC is 12 ng/μL per cell in ~13 μL final volume:
Whole genome amplification product from the C1™ System is used to prepare a target-specific sequencing
library on the Access Array™ System. In this step, target-specific assays are used to enrich the genomic regions of interest for next-generation sequencing.
Polymerization
2
Primer Annealing
1 3
Phi29 Debranching
Primer Annealing
4
5
Polymerization
6
Phi29 Debranching
MULTIPLE DISPLACEMENT AMPLIFICATION
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Revision History
Revision Date Description of change
B1 4 April 2014 Added new content on thawing cells.
A1 4 December 2013
Released new document on use of the Fluidigm C1Single-Cell Auto Prep System with the C 1™ Single-CellAuto Prep Reagent Kit for DNA Seq to perform targeted sequencing of genomic DNA from single cells.
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Whole Genome Amplification and Targeted Sequencing of Single Cells by DNA SeqD
ay 1
: C
1TM IF
C R
un
Pipet riming olutions into IFC
Thaw eagents 5 minutes
QC arvest( ico reen )
0 minutes
Prepare eagent re- ixes 15 minutes
Harvest m from IFC
0 minutes. Store amplicons at 4 °C until use on a 48.48 Access Array IFC
Pipet ysis, top uffer, eaction- nzyme Mix, and arvest eagents into Chip
5 minutes
Image ells with icroscope
5 minutes
10 minutes
5 minutes
Prime IFC on C1 Single-Cell Auto Prep System
Pipet ells into IFC
Load cells on C1 Single-Cell Auto Prep System
15 minutesPrepare reagent pre-mixes, samples, and assays
5 minutesPipet samples assays into IFC
10 minutesPipet arvest eagents
Harvest amplicons from A A IFC
. (Can be run overnight with a pause between DNA Seq and Harvest functions )
: You have up to 60 minutes after the script finishes to remove the harvest amplicons from the IFC.
Run DNA Seq script on the C1 System
Begin loading the second IFC 1 hour after thermal cycling begins on the first IFC.
5 minutes
60 minutesLoad on AA pre-PCRIFC controller
Harvest amplicons from IFC on AA post PCR AX IFC controller
Pipet into
Prime AA IFC on pre-PCR AX IFC controller
2.5 hoursThermocycle AA
Day
2: F
luid
igm
® A
cces
s Ar
ray
R
uns
Reagent Handling Automated Steps Time Estimates
Day 1
C1 IFC Run
Day 2
AA IFC Runs
Day 3
Barcoding & Sequencing
QC ( )
0 minutes
minutes
60 minutes
0 minutes
1 minutes
.
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Figure 1 Whole genome amplification and targeted sequencing workflow.
References
Fluidigm® C1™ Single-Cell Auto Prep System User Guide (PN 100-4977)
LIVE/DEAD® Viability/Cytotoxicity Kit, for mammalian cells (Life Technologies, PN L-3224)
Minimum Specifications for Single-Cell Imaging (Fluidigm, PN 100-5004)
INCYTO Disposable Hemocytometer, www.incyto.com/product/product02_detail.php
Single-Cell WTA PicoGreen Template (Fluidigm, PN 100-6260)
Agilent Bioanalyzer user guide
Illumina® Nextera® Rapid Capture Guide
Voet, T. et al. “Single-cell paired-end genome sequencing reveals structural variation per cell cycle.” Nucleic Acids Res. 41 (2013): 6119-38.
Dean, F. B. et al. “Comprehensive human genome amplification using multiple displacement amplification.” PNAS 99(2002): 5261-66.
Day
3: B
arco
ding
and
Seq
uenc
ing Prepare barcoding reaction
(96-well plate) 30 minutes
50 minutesRun barcoding reaction(stand alone plate thermocycler)
Pool and clean up products 60 minutes
30 minutesQC on bioanalyzer
Sequencing
Continued
Reagent Handling Automated Steps Time Estimates
Day 1C1 IFC
Run
Day 2
AA IFC Runs
Day 3
Barcoding & Sequencing
Targeted Sequencing of Single Cells
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Required Consumables
Required Consumables for Whole Genome Amplification
Required Consumables for Targeted Sequencing
For more information on required consumables after library preparation, refer to the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
Product Name Company Part Number
C1 Single-Cell Auto Prep IFC for DNA Seq (5-10 μm)
C1 Single-Cell Auto Prep IFC for DNA Seq (10-17 μm)
C1 Single-Cell Auto Prep IFC for DNA Seq (17-25 μm)
Fluidigm 100-5762
100-5763
100-5764a
a. See “IFC Types and Related Scripts” on page 11.
VWR® Slick Disposable Microcentrifuge Tubes, Polypropylene, 1.5-mL VWR 20170-666
TempPlate™ semi-skirted 96-well PCR plates USA Scientific 1402-9700
MicroAmp® Clear Adhesive Film Life Technologies 4306311
15-mL centrifuge tubes Major Laboratory Supplier —
Product Name Company Part Number
48.48 Access Array IFC
NOTE: Two IFCs required to process 96 cells
Fluidigm AA-M-48.48
Agilent DNA 1000 Kit Agilent Technologies 5067-1504
Eppendorf® 0.2-mL PCR Tubes VWR 47730-598
VWR® Slick Disposable Microcentrifuge Tubes, Polypropylene, 1.5-mL VWR 20170-666
TempPlate™ semi-skirted 96-well PCR plates USA Scientific 1402-9700
MicroAmp® Clear Adhesive Film Life Technologies 4306311
Corning® 384 Well Low Flange Black Flat Bottom Polystyrene Not Treated Microplate
Corning 3573
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Suggested Consumables
Suggested Consumables for Targeted Sequencing
Required Reagents
Required Reagents for Whole Genome AmplificationSee a diagram of the C1
™ Single-Cell Auto Prep Reagent Kit for DNA Seq in “Appendix 2: C1 Single-Cell Auto Prep Reagent Kit for DNA Seq (PN 100-7357)” on page 43.
Product Name Company Part Number
Barrier Tape Fluidigm PN 100-5920
Product Name Company
C1 Single-Cell Auto Prep
Reagent Kit for DNA Seq(PN 100-7357)
DNA Seq (Module 1):
• C1™ Suspension Reagent
• C1™ Blocking Reagent
• C1™ DNA Dilution Reagent
Fluidigm
DNA Seq (Module 2):
• C1™ Harvest Reagent
• C1™ Preloading Reagent
• C1™ DNA Seq Cell Wash Buffer
• C1™ MgCl2 for AA (Access Array™ System)
DNA Seq (Module 3):
• PCR Water• C1
™ DTT• C1
™ DNA Seq Reaction Mix• C1
™ DNA Seq Lysis Buffer• C1
™ DNA Seq Stop Buffer
illustra GenomiPhi V2 DNA Amplification Kit
GE Healthcare Life Sciences 25-6600-30a
25-6600-31b
a. ~5 chips with tube controls or ~8 chips with no tube controls.b. ~22 chips with tube controls or ~30 chips without tube controls.
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Required Reagents for Targeted Sequencing
For more information on equipment required for targeted sequencing, refer to the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
Suggested Reagents
Suggested Reagents for Whole Genome Amplification
Product Name Company Part Number
48.48 Access Array™ Loading Reagent Kit Fluidigm 100-1032
20X Access Array™ Loading Reagent Fluidigm 100-7604
Access Array™ Barcode Library for Illumina Sequencers - 384 (Single Direction)
Fluidigm 100-4876
FastStart High Fidelity PCR System, dNTPack Roche 04 738 292 001 (500 U)
Agencourt® AMPure® XP Beckman Coulter A63880
Agilent DNA 1000 Kit Agilent Technologies 5067-1504
Order the appropriate primers:
• Custom uniplex primers or• Custom multiplex primers
Fluidigm vendor
Fluidigm vendor
ASY-AA
ASY-AAX
Product Name Company Part Number
LIVE/DEAD Viability/Cytotoxicity Kit Life Technologies L-3224
Quant-IT™ PicoGreen® dsDNA Assay Kit Life Technologies P11496
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Storage Conditions
Whole Genome Amplification ReagentsStored at –20 °C
C1 Harvest Reagent (Fluidigm, PN 100-6248)
C1 Preloading Reagent (Fluidigm, PN 100-5311)
C1 DNA Seq Cell Wash Buffer (Fluidigm, PN 100-7158)
C1 MgCl2 for AA (Fluidigm, PN 100-7249)
PCR Water (Fluidigm, PN 100-5941)
C1 DTT (Fluidigm, PN 100-5873)
C1 DNA Seq Reaction Mix (Fluidigm, PN 100-7154)
C1 DNA Seq Lysis Buffer (Fluidigm, PN 100-7156)
C1 DNA Seq Stop Buffer (Fluidigm, PN 100-7157)
Stored at 4 ºC
C1 Suspension Reagent (Fluidigm, PN 100-5315)
C1 Blocking Reagent (Fluidigm, PN 100-5316)
C1 DNA Dilution Reagent (Fluidigm, PN 100-5317)
Stored at room temperature
70% Ethanol in a squirt bottle
Targeted Sequencing ReagentsSee “Required Reagents for Targeted Sequencing” on page 8, then follow to the manufacturer’s storage recommendations.
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Required Equipment
Required Equipment for Whole Genome Amplification
C1 Single-Cell Auto Prep System
2 centrifuges: 1 for 1.5-mL microcentrifuge tubes, 1 for 96-well plates and 15-mL centrifuge tubes
Vortexer
Thermal cycler
Water bath (37 °C; to thaw frozen cells)
Low-lint cloth
2 biocontainment hoods to prevent DNA contamination of lab and samples
Required Equipment for Targeted Sequencing
Access Array System
Agilent Bioanalyzer
Vortexer
Centrifuge
Thermal Cycler
Magnetic stand for 1.5-mL microcentrifuge tubes
Fluorometer
For more information on equipment required for targeted sequencing, refer to the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
Suggested Equipment
Suggested Equipment for Whole Genome Amplification
INCYTO C-Chip Disposable Hemocytometer (Neubauer Improved, PN DHC-N01)
Imaging equipment compatible with C1 Auto Prep Array integrated fluidic circuits (IFCs). See Minimum Specifications for Single-Cell Imaging Specification Sheet, PN 100-5004.
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IFC Types and Related Scripts
There are currently three C1 System-compatible IFCs for small, medium and large single cells:
Table 1 IFC types and related scripts.
Cell Size(Median)
Barcode (prefix) Script Names Description
Small (5-10 μm)
C1 Single-Cell Auto Prep IFC for DNA Seq (5-10 μm) PN 100-5762
1791x
DNA Seq: Prime (1791x)
Priming the control line and cell capture channels of the 5–10 μm IFC (1791x)
DNA Seq: Cell Load (1791x)
Cell loading and washing without staining for DNA Seq of 5-10 μm diameter cells (1791x)
DNA Seq: Cell Load & Stain (1791x)
Cell loading, staining, and washing for DNA Seq of 5-10 μm diameter cells (1791x)
DNA Seq: Amplify (1791x)
Loading, thermal, and harvest protocol for single-cell lysis and whole genome amplification of 5–10 μm diameter cells (1791x)
Medium (10-17 μm)
C1 Single-Cell Auto Prep IFC for DNA Seq (10-17 μm) PN 100-5763
1792x
DNA Seq: Prime (1792x)
Priming the control line and cell capture channels of the 10-17 μm IFC (1792x)
DNA Seq: Cell Load (1792x)
Cell loading and washing without staining for DNA Seq of 10-17 μm diameter cells (1792x)
DNA Seq: Cell Load & Stain (1792x)
Cell loading, staining, and washing for DNA Seq of 10-17 μm diameter cells (1792x)
DNA Seq: Amplify (1792x)
Loading, thermal, and harvest protocol for single-cell lysis and whole genome amplification of 10-17 μm diameter cells (1792x)
Large (17-25 μm)
C1 Single-Cell Auto Prep IFC for DNA Seq (17-25 μm) PN 100-5764
1793x
DNA Seq: Prime (1793x)
Priming the control line and cell capture channels of the 17-25 μm IFC (1793x)
DNA Seq: Cell Load (1793x)
Cell loading and washing without staining for DNA Seq of 17-25 μm diameter cells (1793x)
DNA Seq: Cell Load & Stain (1793x)
Cell loading, staining, and washing for DNA Seq of 17-25 μm diameter cells (1793x)
DNA Seq: Amplify (1793x)
Loading, thermal, and harvest protocol for single-cell lysis and whole genome amplification of 17-25 μm diameter cells (1793x)
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Safety
It is the individual’s responsibility to review all SDSs for chemicals used in this procedure before running the test (go to:www.fluidigm.com).As with all procedures, these general safety guidelines apply:
Personal Protective Equipment (PPE): safety glasses, fully-enclosed shoes, gloves.
Know the locations of all safety equipment (fire extinguishers, spill kits, eyewashes/showers, first aid kits, safety data sheets, etc.), emergency exit locations, and emergency/injury reporting procedures.
No eating, drinking, or smoking in lab areas.
Maintain clean work areas.
Wash hands before leaving the lab.
Best Practices
• Use good laboratory practices to minimize contamination of samples. Use a new pipette tip for every new sample. Whenever possible, separate pre- and post-PCR activities. Dedicate laboratory materials to designated areas.
HOT SURFACE! The C1 Single-Cell Auto Prep System thermal cycler chuck gets hot and can burn your skin. Use caution when working near the chuck.
PINCH HAZARD! The C1 Single-Cell Auto Prep System door and shuttle can pinch your hand. Make sure your fingers, hand, shirt sleeve, etc. are clear of the door and shuttle when loading or ejecting an IFC.
BIOHAZARD! If you are putting live cells on the C1 Single-Cell Auto Prep System, use the appropriate personal protective equipment and follow your lab’s safety protocol to limit biohazard risks.
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Preparation for Cell Capture and Amplification
Table 2 Cell and DNA seq preparation.
Required Reagents Preparation Kit Name
1.
Prime the IFC
C1 Preloading Reagent Remove from –20 ºC and thaw to room temperature in a DNA-free hood
Fluidigm C1 Kit
C1 Harvest Reagent Remove from -20 ºC and thaw to room temperature in a DNA-free hood
Fluidigm C1 Kit
C1 Blocking Reagent Remove from 4 ºC and keep at room temperature in a DNA-free hood Fluidigm C1 Kit
C1 DNA Seq Cell Wash Buffer Remove from -20 ºC and thaw to room temperature in a DNA-free hood Fluidigm C1 Kit
2.Prepare the (Optional) LIVE/DEAD Cell Staining
Ethidium homodimer-1 Remove from -20 ºC and keep in the dark as much as possible
LIVE/DEAD Kit, Life Technologies
Calcein AM Remove from -20 ºC and keep in the dark as much as possible
LIVE/DEAD Kit, Life Technologies
3.Prepare the Cell Mix
C1 Suspension Reagent Remove from 4 ºC and keep at room temperature in a DNA-free hood Fluidigm C1 Kit
4.Obtain the C1 DNA Stop Buffer
C1 DNA Stop Buffer a
a.Note that the C1 DNA Seq Stop Buffer in the kit has a blue cap but no “S” on the cap.
Remove from -20 ºC, thaw on ice, and keep on ice Fluidigm C1 Kit
5.Prepare the DTT Mix
PCR Water Remove from -20 ºC, and keep at room temperature Fluidigm C1 Kit
GE Kit Sample BufferRemove from -20 ºC, thaw on ice, and keep on ice GE Healthcare Life
Sciences, illustra GenomiPhi V2 DNA Amplification Kit
GE Kit Reaction BufferRemove from -20 ºC thaw on ice, and keep on ice GE Healthcare Life
Sciences, illustra GenomiPhi V2 DNA Amplification Kit
C1 DTT (1 M) Remove only 1 tube from -20 ºC and thaw on ice.
Keep all other tubes frozen. Thaw a new tube only as needed.
Fluidigm C1 Kit
6.Prepare the Lysis Mix
C1 DNA Seq Lysis Buffer Remove from -20 ºC and thaw to room temperature in a DNA-free hood Fluidigm C1 Kit
7.Prepare the Reaction-Enzyme Mix
C1 DNA Seq Reaction Mix
Remove from -20 ºC and thaw to room temperature in a DNA-free hood Fluidigm C1 Kit
GE Kit Enzyme Mix Remove from -80 ºC and vortex well, and keep on iceGE Healthcare Life Sciences, illustra GenomiPhi V2 DNA Amplification Kit
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Preparation for DNA Sequencing
Required Reagents Preparation Kit Name
8.Perform Access Array™ for Targeted Sequencing
FastStart High Fidelity PCR System, dNTPack Remove from -20 ºC, thaw on ice, and keep on ice Roche, PN 04-738-
292-00120X Access Array™ Loading Reagent Remove from -20 ºC, thaw on ice, and keep on ice Fluidigm, PN 100-
08831X Access Array™ Harvest Solution Remove from -20 ºC, thaw on ice, and keep on ice Fluidigm, PN 100-
1031
Access Array™ Barcode Library for Illumina Sequencers - 384 Remove from -20 ºC, thaw on ice, and keep on ice
Fluidigm, PN 100-4876
C1 DNA Dilution Reagent Remove from 4 ºC, thaw on ice, and keep on ice Fluidigm C1 Kit
Custom uniplex or multiplex primers Remove from -20 ºC, thaw on ice, and keep on ice Fluidigm, PN ASY-
AAX20X primer solution preparationa
a. Refer to the Fluidigm® Access Array™ Multiplex 20X Primer Solution Preparation Quick Reference (PN 100-3895).
Remove from -20 ºC, thaw on ice, and keep on ice Primers customer-supplied
PCR Water Remove from -20 ºC, and keep at room temperature Fluidigm C1 Kit
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Use the Chip Map Loading Plate
A black Chip Map Loading Plate accessory can be used to assist IFC (chip) pipetting.
1 Obtain a Chip Map Loading Plate:
Figure 2 Chip Map Loading Plate. Note that the C1™ DNA Seq Stop Buffer in the kit has a blue cap but no “S” on the cap.
2 Place the C1 Single-Cell Auto Prep IFC onto the Chip Map Loading Plate. For details on IFC (chip) loading, see “Appendix 1: IFC (Chip) Pipetting Map” on page 42.
3 Prime the IFC (see “Prime the IFC” on page 16).
NOTE: The Chip Map Loading Plate is supplied with the C1 Single-Cell Auto Prep System. If you do not have a Chip Map Loading Plate, contact Fluidigm Technical Support.
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Prime the IFC
Figure 3 C1™ IFC priming pipetting map.
1 Pipet 200 μL of C1 Harvest Reagent from 4 mL bottle into accumulators marked with red circles in Figure 3.
2 Pipet 20 μL of C1 Harvest Reagent into wells marked with red squares on each side of the accumulators (36 total).
3 Pipet 20 μL of C1 Harvest Reagent into the two wells on each side of the IFC marked with red squares. These wells are marked on the bottom of the IFC with a notch to ensure they are easily located.
4 Pipet 20 μL of C1 Preloading Reagent into Inlet 2 and Inlet 5, both marked with a purple dot. 5 Pipet 15 μL of C1 Blocking Reagent into the Cell Inlet and Outlet marked with white dots.6 Pipet 20 μL of C1 DNA Seq Cell Wash Buffer, marked with a blue dot, into Inlet 3.7 Peel off white tape on bottom of IFC.
NOTE: When pipetting into the C1 IFC, always stop at the first stop on the pipette to avoid creating bubbles in the inlets. If a bubble is introduced, ensure that it floats to the top of the well.
NOTE: Vortex and spin all reagent mixes before pipetting into the IFC.
IMPORTANT: Allow C1 DNA Seq Cell Wash Buffer (Fluidigm) to equilibrate to room temperature prior to use.
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8 Place the IFC into the C1 Single-Cell Auto Prep System then run the DNA Seq: Prime (1791x/1792x/1793x) script. Priming takes approximately 10 minutes. When the Prime script has finished, tap EJECT to remove the primed IFC from the instrument.
Prepare Cells
• “(Optional) Prepare LIVE/DEAD Cell Staining Solution” on page 17• “Prepare the Cells” on page 18• “Prepare the Cell Mix” on page 20• “Load the Cells” on page 21• “Image the Cells” on page 22
(Optional) Prepare LIVE/DEAD Cell Staining SolutionThe optional live/dead cell staining step uses the LIVE/DEAD Viability/Cytotoxicity Kit, which tests the viability of a cell based on the integrity of the cell membrane. This test contains two chemical dyes. The first dye is green-fluorescent calcein-AM, which stains live cells. This dye is cell-permeable and tests for active esterase activity in live cells. The second dye is red-fluorescent ethidium homodimer-1, which will stain nucleic acids only if the integrity of the cell membrane has been lost.
NOTE: After priming the IFC, you have 1 h to load the cells on the IFC in the C1 Single-Cell Auto Prep System.
IMPORTANT: •Allow C1 DNA Seq Cell Wash Buffer (Fluidigm) to equilibrate to room temperature prior to use.
•Use the mixes 30 minutes after preparation.
NOTE: Keep the dye tubes closed and in the dark as much as possible, as they can hydrolyze over time. When not in use, store in dark, airtight bag with desiccant pack at -20 ºC.
NOTE: Cell staining solution may be prepared up to 2 hours before loading onto the C1 IFC. Keep on ice and away from light before pipetting into IFC.
IMPORTANT: Staining small cells (5–10 μm) takes 30 minutes, and staining medium (10–17 μm) or large (17–25 μm) cells takes 60 minutes. To easily visualize small cells, you may need to double the amount of stain used.
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1 Vortex the dyes well before pipetting. 2 Prepare the LIVE/DEAD stain:
3 Vortex the C1 LIVE/DEAD staining solution before pipetting onto an IFC.
Prepare the Cells(Optional) Thaw the cells
If you do not need to thaw the cells, proceed to “Wash the Cells.”
1 Warm 10 mL of the appropriate cell culture medium to 37 °C.2 Thaw 1 mL of cells in a 37 °C water bath: For <1 minute, shake the tube of cells in the water
bath to steadily increase the temperature of the cell suspension.3 Transfer the entire thawed cell suspension to a 15-mL centrifuge tube.4 Slowly add 9 mL the warmed cell culture media drop-wise.5 Close the 15-mL centrifuge tube, then gently invert the tube 2–3 times.6 Centrifuge the cells at 300 x g for 5 minutes, then remove and discard the supernatant.7 Gently resuspend the cell pellet in 1 mL of C1 DNA Seq Cell Wash Buffer.
Wash the Cells
1 Transfer 1 mL of the cell suspension to a new tube.2 Centrifuge to pellet the cells at 300 x g for 5 minutes.3 Remove 900 μL of supernatant.4 Add 900 μL of C1 DNA Seq Cell Wash Buffer, then gently pipet the cells up and down >5 times.
Components Volume (μL)
C1 DNA Seq Cell Wash Buffer (Fluidigm)(26-mL bottle) 1250
Ethidium homodimer-1 (LIVE/DEAD kit, Life Technologies) 2.5
Calcein AM (LIVE/DEAD kit, Life Technologies) 0.625
Total 1253.125
IMPORTANT: The thawing procedure is stressful to frozen cells. Using good technique and working quickly ensures that a high proportion of the cells survive the procedure.
IMPORTANT: The whole genome amplification protocol amplifies all DNA in the reaction. You must remove as much debris from the cells as possible. Washing the cells before loading them on the IFC is required.
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5 Centrifuge the cells at 300 x g for 5 minutes.
6 Remove 900 μL of supernatant.
7 Add 900 μL of C1 DNA Seq Cell Wash Buffer, then gently pipet the cells up and down >5 times.8 Repeat washing the cells:
• Fresh cells: Repeat steps 5–7 one time.• Thawed cells from frozen sample: Repeat steps 5–7 three times.
9 Count the cells, then dilute the cells with C1 DNA Seq Cell Wash Buffer to 166–250 cells/μL.Prepare a Cell Suspension
Prepare a cell suspension at a concentration of 166–250 cells/μL in C1 DNA Seq Cell Wash Buffer prior to mixing with C1 Cell Suspension Reagent and loading onto the IFC. This will ensure a total cell count pipetted on the IFC of approximately 500–750 cells. As few as 200 cells total, from 66 K/mL in native medium, may be loaded on the IFC. Fewer cells loaded may yield fewer captured cells. A final volume of 0.5–1 mL is desirable so that there are enough cells for both the C1 IFC and the tube controls.
IMPORTANT: For the last wash of thawed cells, centrifuge the cells at 100–200 x g.
IMPORTANT: For the last wash of thawed cells, save the supernatant should you need to recover additional cells. Recover more cells by centrifuging the saved supernatant at 500 x g.
NOTE: Cells may be counted by any preferred method. If an established cell counting protocol does not exist, we suggest using the disposable hemocytometer C-Chip by INCYTO. See www.incyto.com for instructions for use.
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Prepare the Cell Mix
1 Ensure that you have begun priming the IFC (see “Prime the IFC” on page 16).2 Prepare Cell Mix by combining cells with C1 Cell Suspension Reagent at a ratio of 3:2. For
example:
3 Set a P200 pipette to 60 μL, then pipet the Cell Mix up and down 5–10 times to mix, depending on whether the cells tend to clump. Do not vortex the cell mix. Avoid bubbles when mixing as these may cause load failures.
IMPORTANT: Vortex the C1 Suspension Reagent thoroughly prior to use. If C1 Suspension Reagent contains particulate, ensure they are properly removed by vortexing. Do NOT vortex the cells.
Components Volume (μL)
166-250 cells/μL 60
C1 Suspension Reagent (Fluidigm) 40
Total 100
NOTE: The volume of Cell Mix may be scaled depending on volume of cells available. A minimum volume of 5 μL of Cell Mix is necessary for the IFC. Maintain a ratio of cells to C1 Suspension Reagent of 3:2.
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Load the Cells
Figure 4 C1™ IFC loading pipetting map.
1 Remove blocking solutions from cell inlet and outlet marked with teal and white dots in Figure 4.
2 Pipet Cell Mix up and down 5–10 times to mix, depending on whether the cells tend to clump. Do not vortex the Cell Mix. Avoid bubbles when mixing, as these may cause load failures.
3 Pipet 5 μL of Cell Mix into the cell inlet marked with the teal dot. You may pipet up to 20 μL of Cell Mix, however only 5 μL will enter the IFC.
4 Perform one of these tasks:• Staining cells: Vortex the C1 LIVE/DEAD staining solution well, then pipet 20 μL of the
solution into inlet 1 marked with a pink dot.• Not staining cells: Pipet 20 μL of C1 DNA Seq Cell Wash Buffer into inlet 1 marked with a pink
dot.5 Place the IFC into the C1 Single-Cell Auto Prep System then run the DNA Seq: Cell Load (1791x/
1792x/1793x) or DNA Seq: Cell Load & Stain (1791x/1792x/1793x) script. If loading only, this will take approximately 30 minutes; if loading and staining, about 60 minutes. When the script has finished tap EJECT to remove the IFC from the C1 Single-Cell Auto Prep System.
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Image the CellsCells may be imaged on a microscope compatible with C1 IFCs. Guidelines for the selection of a microscope are outlined in Minimum Specifications for Single-Cell Imaging, PN 100-5004.
Prepare Reagent Mixes
• “Obtain C1 DNA Seq Stop Buffer”• “Prepare the DTT Mix” on page 23• “Prepare the Lysis Mix” on page 23• “Prepare the Reaction-Enzyme Mix” on page 24
Obtain C1 DNA Seq Stop BufferC1 DNA Seq Stop Buffer is ready to use.
Vortex the solution for 5 seconds, then centrifuge it to collect the buffer. Keep on ice until use.
NOTE: Vortex and spin all reagent mixes before pipetting into the IFC.
IMPORTANT: Use the mixes 30 minutes after preparation as described in the next sections.
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Prepare the DTT Mix
1 Label a new tube (suggestions: “DTT Mix” or “D”).2 Mix the reagents in the labeled tube:
3 Vortex the solution for 5 seconds, then centrifuge it to collect the mix. Keep on ice until use.
Prepare the Lysis Mix1 Label a new tube (suggestions: “Lysis Mix” or “L”).2 Mix the reagents in the new labeled tube:
3 Vortex the solution for 5 seconds, then centrifuge it to collect the mix. Keep on ice until use.
IMPORTANT: Remove only 1 tube of C1 DTT from -20 ºC and thaw on ice. Keep all other tubes
frozen. Thaw a new tube only as needed.
Components Volume (μL)
PCR Water 193.10
GE Kit Sample Buffer 2.30
GE Kit Reaction Buffer 2.30
C1 DTT (1 M; Fluidigm) 2.30
Total 200.00
Components Volume for One C1 IFC (μL)
Volume Including Tube Controls
(μL)a
a. See “Prepare the DNA Seq Reaction tube controls” on page 28.
C1 DNA Seq Lysis Buffer (Fluidigm) 13.5 19.8
C1 DTT (1 M; Fluidigm) 1.5 2.2
Total 15.0 22.0
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Prepare the Reaction-Enzyme Mix
1 Label a new tube (suggestions: “Reaction-Enzyme Mix” or “RE”). 2 Mix the reagents in the labeled tube:
3 Vortex the solution for 5 seconds, then centrifuge it to collect the mix. Keep on ice until use.
IMPORTANT: Use DTT Mix previously prepared (see “Prepare the DTT Mix” on page 23), not stock DTT solution. Use C1 DNA Seq Reaction Buffer, not GE Reaction Buffer.
Components Volume for One C1 IFC (μL)
Volume Including Tube Controls
(μL)a
a. See “Prepare the DNA Seq Reaction tube controls” on page 28.
C1 DNA Seq Reaction Mix 30.0 45.00
GE Kit Enzyme Mix 3.0 4.50
DTT Mix (see “Prepare the DTT Mix” on page 23) 21.0 31.50
Total 54.00 81.00
NOTE: Discard the remaining DTT Mix. Do not reuse. Prepare fresh mix as needed.
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Run Lysis and DNA Amplification on the C1 System
Figure 5 C1 IFC Lysis, neutralization, and DNA Seq pipetting map. Note that the C1 DNA Seq Stop Buffer in the kit has a blue cap but no “S” on the cap.
1 Pipet 180 μL of C1 Harvest Reagent into the four reservoirs marked with large solid red
rectangles in Figure 5.2 Pipet 10 μL of Lysis Mix in Inlet #4 with a light tan dot.3 Pipet 10 μL of C1 DNA Seq Stop Buffer in Inlet #6 with a dark blue dot (“S”).4 Pipet 24 μL of Reaction-Enzyme Mix in Inlets #7 and #8 marked with light green dots.
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5 Place the IFC into the C1 Single-Cell Auto Prep System and run the DNA Seq: Amplify (1791x/1792x/1793x) script.
NOTE: The DNA Seq: Amplify (1791x/1792x/1793x) script may be run overnight. Approximate run times are:
• Small-cell IFC: ~6.25 hours (~5.0 hours for lysis and amplification; and 1.25 hoursfor harvest)
• Medium- and large-cell IFCs: ~7.5 hours (~5.0 hours for lysis and amplification;and ~2.5 hours for harvest)
This protocol can be programmed to harvest at a convenient time. Slide the orange box (end time) to the desired time. For example, the harvest function could be programmed to next morning:
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The DNA Seq: Amplify (1791x/1792x/1793x) script contains these thermal cycling protocols:
(Optional) Prepare the Tube Controls
The tube controls are used as positive and negative controls for the DNA Seq workflow. You prepare three tube controls: the Lysis Reaction, Stop Lysis Reaction, and DNA Seq Tube Controls. For each control, you prepare three solutions containing gDNA (Tube 1), cells (Tube 2), or PCR Water (Tube 3; no template control or NTC).
Prepare the Lysis Reaction Tube Controls1 Add Lysis Mix (see “Prepare the Lysis Mix” on page 23) to gDNA (GE Healthcare Life Sciences),
cells, and PCR Water in new tubes:
2 Vortex the solutions for 5 seconds, then centrifuge it to collect the mix.3 Place the Lysis Reaction tube controls on ice for 10 minutes, then immediately proceed to
“Prepare the Stop Lysis Reaction Tube Controls” on page 28:
Lysis DNA Seq
Temperature Time (min) Temperature Time (min)
4 ºC 10 38 ºC 120
25 ºC 1 70 ºC 15
Stop Lysis
Temperature Time (min)
23 ºC 3
Components Tube 1: gDNA(μL)
Tube 2: Cells (μL)
Tube 3: NTC (μL)
gDNA, GE Kit Control DNA, 10 ng/μL 1.0 — —
Cell Mix — 1.0 —
PCR Water — — 1.0
Lysis Mix 2.0 2.0 2.0
Total 3.00 3.00 3.00
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Prepare the Stop Lysis Reaction Tube Controls1 Add C1 DNA Seq Stop Buffer (Fluidigm) to the same tubes containing the Lysis Reaction tube
controls (see “Prepare the Lysis Reaction Tube Controls” on page 27):
2 Vortex the solutions for 5 seconds, then centrifuge it to collect the mix.3 Incubate the Stop Lysis Reaction tube controls at room temperature for 3 minutes, then keep
the controls at room temperature.
Prepare the DNA Seq Reaction tube controls1 Add Reaction-Enzyme Mix (see “Prepare the Reaction-Enzyme Mix” on page 24) to aliquots of
Stop Lysis Reaction (see “Prepare the Stop Lysis Reaction Tube Controls”) in new tubes:
2 Vortex the solutions for 5 seconds, then centrifuge the tubes to collect the mix.3 Place the tubes in a thermal cycler and run the following program:
Components Tube 1: gDNA(μL)
Tube 2: Cells (μL)
Tube 3: NTC (μL)
Lysis Reaction 3.0 3.0 3.0
C1 DNA Seq Stop Buffera
a. Note that the C1 DNA Seq Stop Buffer in the kit has a blue cap but no“S” on the cap.
4.0 4.0 4.0
Total 7.00 7.00 7.00
Components Tube 1: gDNA(μL)
Tube 2: Cells (μL)
Tube 3: NTC (μL)
Stop Lysis Reaction 1.05 1.05 1.05
Reaction-Enzyme Mix 8.95 8.95 8.95
Total 10.00 10.00 10.00
Temperature Time (min)
38 ºC 120
70 ºC 15
4 ºC Hold
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4 Dilute the three amplified tube controls:
a Transfer prepared material to a post-PCR room.
b Vortex the solution for 5 seconds, then centrifuge it to collect the mix.
c Combine:
5 Quantify the tube controls with the PicoGreen dsDNA quantitation assay to perform quality control on the protocol (see “Quantify Harvest Amplicons”).
Harvest the Amplified DNA Products
1 When the DNA Seq: Amplify script has finished, tap EJECT to remove the IFC from the instrument.
2 Transfer the C1 IFC to a post-PCR lab environment.3 Label a new 96-well plate “DILUTED HARVEST PLATE.”4 Aliquot 10 μL of C1 DNA Dilution Reagent into each well of the DILUTED HARVEST PLATE.5 Carefully pull back the tape covering the harvesting inlets of the IFC using the plastic removal
tool:
Figure 6 Tape removal.
Components Volume (μL)
C1 DNA Dilution Reagent (Fluidigm) 9
PCR Products 1
Total 10
NOTE: The IFC may remain in the C1 Single-Cell Auto Prep System for up to one hour after harvest before removing products from their inlets.
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6 Use an 8-channel pipette to pipet the harvested amplicons from the inlets according to the diagram (see Figure 7) and table (see Table 3) and place in the DILUTED HARVEST PLATE:
Figure 7 Pipette map of reaction products on the C1 IFC
Table 3 Harvest amplicon dilution
NOTE: Harvest volumes may vary. Set a pipette to 4.0 μL to ensure entire volume is extracted.
Components Volume (μL)
C1 DNA Dilution Reagent (Fluidigm) 10
C1 harvest amplicons ~3.5
Total ~13.5
NOTE: For detailed instructions on pipetting the harvested aliquots to the “DILUTED HARVEST PLATE,” proceed to steps 7–10.
1 2 3 7 8 9 131415
4 5 6 10 11 12 16 17 18
19 20 21 25 26 27 31 32 33
22 23 24 28 29 30 34 35 36
37 38 39 43 44 45 49 50 51
40 41 42 46 47 48 52 53 54
55 56 57 61 62 63 67 68 69
58 59 60 64 65 66 70 71 72
73 74 75 79 80 81 85 86 87
76 77 78 82 83 84 88 89 90
91 92 93 94 95 96
4 5 610 11 1216 17 1822 23 2428 29 3034 35 36
40 41 4246 47 4852 53 54
58 59 6064 65 6670 71 7276 77 7882 83 8488 89 90
94 95 96
1 2 37 8 9 13141519 20 2125 26 2731 32 33
37 38 3943 44 4549 50 5155 56 5761 62 6367 68 6973 74 7579 80 8185 86 8791 92 93
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7 Pipet the entire volume of C1 harvest amplicons out of the left-side wells of the C1 IFC into the 10 μL of C1 DNA Dilution Reagent in each well of the DILUTED HARVEST PLATE:
Figure 8 First three harvest product pipette steps
8 Pipet the entire volume of C1 harvest amplicons out of the right-side wells of the C1 IFC into the 10 μL of C1 DNA Dilution Reagent in each well of the DILUTED HARVEST PLATE:
Figure 9 Fourth, fifth, and sixth pipetting steps
LEFT side 96-well plate
1 2 3
7 8 9
13 14 15
19 20 21
25 26 27
31 32 33
37 38 39
43 44 45
49 50 51
55 56 57
61 62 63
67 68 69
73 74 75
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85 86 87
91 92 93
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1 2 3 4 5 6 7 8 9 10 11 12
RIGHT side 96-well plate
1 2 3 4 5 6 7 8 9 10 11 125 6
10 11 12
17 18
22 23 24
29 30
34 35 36
41 42
46 47 48
53 54
58 59 60
65 66
70 71 72
77 78
82 83 84
89 90
94 95 96
4
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9 Pipet the entire volume of C1 harvest amplicons out of the left-side wells of the C1 IFC into the 10 μL of C1 DNA Dilution Reagent in each well of the DILUTED HARVEST PLATE:
Figure 10 Seventh, eighth, and ninth pipetting steps
10 Pipet the entire volume of C1 harvest amplicons out of the right-side wells of the C1 IFC into the 10 μL of C1 DNA Dilution Reagent in each well of the DILUTED HARVEST PLATE:
Figure 11 Tenth, eleventh, and twelfth pipetting steps
LEFT side 96-well plate
1 2 3 4 5 6 7 8 9 10 11 121 2 3
7 8 9
13 14 15
19 20 21
25 26 27
31 32 33
37 38 39
43 44 45
49 50 51
55 56 57
61 62 63
67 68 69
73 74 75
79 80 81
85 86 87
91 92 93
8
20
32
44
56
68
80
92
9
21
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45
57
69
81
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RIGHT side 96-well plate
1 2 3 4 5 6 7 8 9 10 11 125 6
10 11 12
17 18
22 23 24
29 30
34 35 36
41 42
46 47 48
53 54
58 59 60
65 66
70 71 72
77 78
82 83 84
89 90
94 95 96
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11
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11 Seal, vortex, and spin down plate.
Quantify Harvest Amplicons
Before proceeding to Access Array Amplification, quantify the harvest amplicons to verify DNA concentrations:
• We suggest using the PicoGreen dsDNA quantitation assay to determine the concentration ofDNA samples; however, alternate methods can be used.
• We suggest using the Microsoft® Excel worksheet, Single-Cell Sequencing PicoGreen® Template(Fluidigm, PN 100-6260), to quantify the library.
• The typical concentration of diluted product is 5-15 ng/μL. If you cannot dilute the product to5-15 ng/μL, contact Fluidigm Technical Support.
NOTE: These samples are now ready for library preparation for sequencing. Samples can be stored for up to 1 week at 4 ºC or at –20 ºC for long-term storage.
NOTE: If you prepared tube controls, you can replace some harvest amplicons with tube controls to perform quality control on the protocol with the PicoGreen® dsDNA quantitation assay [see “(Optional) Prepare the Tube Controls” on page 27].
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Targeted Sequencing
Follow these protocols to perform targeted sequencing:• “Targeted Sequencing: Perform Access Array Amplification”• “Targeted Sequencing: Prepare Barcoded Libraries for Sequencing by the Illumina System” on
page 41
Targeted Sequencing: Perform Access Array Amplification
WorkflowTo review the workflow for targeted sequencing, see “Whole Genome Amplification and Targeted Sequencing of Single Cells by DNA Seq” on page 4.
Required Materials
Required Reagents Stored at –20 ºC• FastStart High Fidelity PCR System, dNTPack (Roche, PN 04-738-292-001)• 20X Access Array™ Loading Reagent (Fluidigm, PN 100-7604)• 1X Access Array™ Harvest Solution (Fluidigm, PN 100-1031)
• C1 MgCl2 for AA (Fluidigm, C1 Single-Cell Auto Prep Reagent Kit for DNA Seq, PN 100-7357)
• PCR Water (Fluidigm, C1 Single-Cell Auto Prep Reagent Kit for DNA Seq, PN 100-7357)
Required Consumables• Two, 48.48 Access Array IFCs (Fluidigm, PN AA-M-48.48)• Eppendorf® 0.2-mL PCR Tubes (VWR, PN 47730-598)• VWR® Slick Disposable Microcentrifuge Tubes, Polypropylene, 1.5-mL (VWR, PN 20170-666)
NOTE: 1X Access Array Harvest Solution (Fluidigm, PN 100-1031) is not packaged for individual sale. It can be purchased in units of 5, under the name Access Array™ Harvest Pack, PN 100-3155 or as a component in the 48.48 Access Array Loading Reagent Kit, PN 100-1032.
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Prime the 48.48 Access Array IFC
1 Inject Control Line Fluid into each accumulator on the IFC [refer to the Fluidigm® Control Line Fluid Loading Procedure Quick Reference (PN 68000132)].
2 Add 500 μL of 1X Access Array Harvest Solution (Fluidigm, PN 100-1031) into the H1-H4 wells on the IFC.
3 Remove and discard the blue protective film from the bottom of the AA IFC.4 Ensure that the IFC Controller AX is located in the pre-PCR lab.5 Tap Eject to move the tray out of the pre-PCR IFC Controller AX.6 Place the IFC onto the tray by aligning the notched corner of the IFC to the A1 mark.7 Tap Load Chip to register the barcode of the IFC and activate the script selection.8 Select Prime (151x) and Run Script to prime the IFC.9 Once the script is complete, tap Eject to remove the IFC.
Figure 12 48.48 Access Array IFC overview
IMPORTANT: •Use the 48.48 Access Array IFC within 24 hours of opening the package.
•Control Line Fluid on the IFC or in the inlets makes the IFC unusable. Useonly 48.48 syringes with 300 μL of Control Line Fluid (Fluidigm, PN 89000020).
•Load the IFC into the Pre-PCR IFC Controller AX in the Pre-PCR lab within 60minutes of priming.
Interface Accum
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Prepare the 20X Primer Solutions
Prepare 20X primer solutions according to your performing uniplex or multiplex PCR:
• Multiplex PCR: Refer to the Fluidigm® Access Array™ Multiplex 20X Primer Solution Preparation Quick Reference (PN 100-3895).
• Uniplex PCR: Refer to “Preparing the 20X Primers Solutions,” in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
IMPORTANT: Warm the 20X Access Array™ Loading Reagent to room temperature before use.
NOTE: The final Tagged TS forward and reverse concentrations for multiplex PCR are 1 μM per primer in the 20X Primer Solutions. The final TS Forward and Reverse Primer concentrations in the Access Array™ reaction chamber are 50 nM per primer.
NOTE: The final TS forward and reverse primer concentrations for uniplex PCR are 4 μM per primer in the 20X Primer Solution. The final TS forward and reverse primer concentrations in the Access Array™ IFC reaction chamber are 200 nM per primer.
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Prepare Sample Master Mix Solutions All DNA samples need to be added into the Sample Pre-Mix individually, prior to loading the Sample Mix solutions into the Sample Inlets of an 48.48 Access Array IFC.
Prepare the Sample Pre-Mix Solutions1 Working in a DNA-free hood, combine the components:
2 Vortex the Sample Pre-Mix for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components.
Prepare the Sample Mix Solutions1 Combine the components listed in a 96-well plate to prepare 48 individual Sample Mix
solutions:
2 Vortex the Sample Mix solutions for a minimum of 20 seconds and centrifuge for 30 seconds to spin down all components.
Component Volume per Reaction (μL)
Volume for 60 Reactions (μL) for one 48.48 Access Array™
IFC
Volume for 120 Reactions (μL) for two 48.48 Access Array™
IFCs
10X FastStart High Fidelity Reaction Buffer without 18 mM MgCl2 (Roche) 0.50 30.0 60.0
C1 MgCl2 for AA (Fluidigm)a
a. Use the MgCl2 provided in the Fluidigm DNA Seq Kit, which is supplied at a higher concentration of MgCl2 thanis provided in the Roche Kit.
0.02 1.4 2.7
DMSO (Roche) 0.25 15.0 30.0
10 mM PCR Grade Nucleotide Mix (Roche) 0.10 6.0 12.0
5 U/μL FastStart High Fidelity Enzyme Blend (Roche) 0.05 3.0 6.0
20X Access Array Loading Reagent (Fluidigm PN 100-0883) 0.25 15.0 30.0
PCR Water (Fluidigm) 0.08 4.6 9.3
Total 1.25 75.0 150.0b
b. This is enough reagent to load two 48.48 Access Array™ IFCs with 24 additional reactions to compensate for dead volume and pipetting error.
Component Volume per Reaction (μL)
Sample Pre-Mix 1.25
DNA Seq diluted harvest 3.75
Total 5.0
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Load the 48.48 Access Array IFC1 Pipet 4 μL of 20X Primer Solution into each of the Primer Inlets.2 Pipet 4 μL of Sample Mix solution into each of the Sample Inlets:
Figure 13 Pipetting order
3 Ensure that the IFC Controller AX is located in the pre-PCR lab.4 Tap Eject to move the tray out of the IFC Controller AX.5 Place the IFC onto the tray by aligning the notched corner of the IFC to the A1 mark.6 Tap Load Chip to register the barcode of the IFC and activate the script selection.7 Select Load Mix (151x) and Run Script.
IMPORTANT: It is essential to vortex all components to ensure complete mixing.
IMPORTANT: While pipetting, do not go past the first stop on the pipette. Doing so may introduce air bubbles into the inlets.
NOTE: An 8-channel pipette is recommended to load the Sample Mix and 20X Primer Solutions. The recommended pipetting order is shown:
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8 After the script is complete, tap Eject to remove the IFC.9 Place the chip onto one of the following thermal cyclers and run the PCR according to the
protocol specified:• Fluidigm FC1™ Cycler: Select the AA 48X48 Standard v1 protocol [refer to the Fluidigm®
FC1™ Cycler Usage Quick Reference, (PN100-1250)].• Fluidigm Stand-Alone Thermal Cycler: Select the AA48v1 protocol [refer to the Fluidigm®
Stand-Alone Thermal Cycler Usage Quick Reference (PN 68000111)].
Harvest the 48.48 Access Array™ IFCFluidigm recommends harvesting the amplicons in a post-PCR lab.
1 After the PCR has finished, move the 48.48 Access Array IFC from the BioMark™ System or BioMark™ HD System into the post-PCR lab for harvesting.
2 Remove the remaining 1X Access Array Harvest Reagent from the H1-H4 wells.3 Pipet 600 μL of fresh 1X Access Array Harvest Reagent into the H1-H4 wells.4 Pipet 2 μL of 1X Access Array Harvest Reagent into each of the Sample Inlets on the IFC.5 Ensure that the IFC Controller AX is located in the post-PCR lab. If a post-PCR lab is not
available, apply barrier tapes to the IFC (see “Appendix 3: How to Use Barrier Tape” on page 44).
6 Tap Eject to move the tray out of the post-PCR IFC Controller AX.7 Place the IFC onto the tray by aligning the notched corner of the IFC to the A1 mark.8 Tap Load Chip to register the barcode of the IFC and activate the script selection.9 Select Harvest (151x) and Run Script.10 Once the script is complete, tap Eject to remove the IFC.11 Label a 96-well plate with the 48.48 Access Array IFC barcode. Carefully transfer 10 μL of
harvested PCR products from each of the Sample Inlets into columns 1-6 of the 96-well PCR plate, using an 8-channel pipette.
Perform Quality Control (QC) on the Harvested PCR productsPerform QC on the harvested PCR products using the Agilent 2100 Bioanalyzer with DNA1000 Chips.
1 Take 1 μL of each of the harvested PCR products in a random sampling of the products, and run each of them on a Bioanalyzer DNA 1000 Chip following the manufacturer’s instructions.
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2 Ensure that amplicon sizes and distribution are within the expected range (+/- 5% for amplicons of 200-400 bp including tags). The primer-dimer contamination in the PCR product pool (in the range of 50–130 bp) should be <25% based on the Bioanalyzer quantification:
Figure 14 DNA 1000 Chip electropherogram of a pooled PCR product with 48 amplicons ranging between 180–350 bp. A. Harvested PCR product pool from a 48.48 Access Array™ IFC. B. The same PCR product pool after cleanup with magnetic beads.
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Targeted Sequencing: Prepare Barcoded Libraries for Sequencing by the Illumina System
To prepare your library for Illumina sequencing:• “Attach Sequence Tags and Sample Barcodes to the Harvested PCR Products”• “Pool, Purify, and Qualify the Barcoded Library”• “Prepare Reagents for Sequencing on the Illumina® Sequencing System”
Attach Sequence Tags and Sample Barcodes to the Harvested PCR Products1 Attach sequence tags and sample barcodes to the harvested PCR products. Refer to “Attaching
Sequence Tags and Sample Barcodes” in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
2 Check the barcoded PCR products on the Agilent 2100 Bioanalyzer. Refer to “Checking PCR Products on the Agilent 2100 BioAnalyzer” in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
Pool, Purify, and Qualify the Barcoded Library1 Pool the barcoded PCR products. Refer to “Pooling Products from Multiple Access Array™ IFCs”
in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
2 Purify the barcoded library. Refer to “Purification of Harvested PCR Products” in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
3 Qualify the barcoded library. Refer to “Agilent 2100 Bioanalyzer Qualification” in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
Prepare Reagents for Sequencing on the Illumina® Sequencing System1 Follow the appropriate protocol to prepare reagents for the sequencing run according to your
Illumina® Sequencing System. Refer to “Preparing Reagents for Sequencing on the Illumina® GAII and HiSeq Sequencing Systems” in the Fluidigm® Access Array™ System for Illumina® Sequencing Systems User Guide (PN 100-3770).
2 Perform the sequencing run on the pooled, barcoded library. Refer to the appropriate Illumina sequencing guide before performing the sequencing run.
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Appendix 1: IFC (Chip) Pipetting Map
#1#2#3#4
#5#6#7#8
A1
S
OutputsOutputs
Key
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Appendix 2: C1 Single-Cell Auto Prep Reagent Kit for DNA Seq (PN 100-7357)
For storage conditions, see “Storage Conditions” on page 9.
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Appendix 3: How to Use Barrier Tape
If you cannot harvest the Access Array IFC in a post-PCR lab, apply barrier tapes (Fluidigm, PN 100-5920) to the IFC. 1 Separate the barrier tape from its base layer and discard the base layer.2 Affix the tape onto the sample inlets before harvesting.3 Ensure that the barrier tape covers all the sample Inlets:
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For More Information
To find out more about the information in this or any other Fluidigm Protocol, email [email protected] or call:
North America: 1 866 358 4354Europe: +33 1 60 92 42 40 Japan: +81 3 3662 2150All other countries: +1 650 266 6100
Fluidigm Product Patent NoticeFluidigm products including IFCs (integrated fluidic circuits/microfluidic chips with or without a carrier)such as C1
™ Single-Cell Auto Prep IFCs, Access Array™ IFCs, Dynamic Array™ IFCs and Digital Array™ IFCs, theIFC controller, FC1™ Cycler and the Fluidigm system (BioMark™ System, BioMark™ HD System, EP1™ System, readers, thermalcycler, etc.) and methods for reading and controlling the C1 Single-Cell Auto Prep IFCs, Access ArrayIFCs, Dynamic Array IFCs and Digital Array IFCs and/or their use and manufacture may be covered by oneor more of the following patents owned by Fluidigm Corporation and/or sold under license fromCalifornia Institute of Technology and other entities: U.S. Patent Nos. 6,408,878, 6,645,432, 6,719,868,6,767,706, 6,793,753, 6,929,030, 7,216,671, 7,323,143, 7,476,363, 7,494,555, 7,601,270, 7,604,965,7,666,361, 7,704,735, 7,749,737, 7,766,055, 7,837,946, 7,867,454, 7,867,763, 8,104,497, 8,104,515,8,105,553, 8,105,824, 8,129,176, 8,157,434, 8,163,492, 8,273,574, 8,257,666; EP Patent Nos. 1065378,1194693, 1195523 and 1345551; and additional issued and pending patents in the U.S. and othercountries. V6.1
For Research Use Only. Not for use in diagnostic procedures.
© 2014 Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, Access Array, BioMark, C1, and Dynamic Array are trademarks or registered trademarks of Fluidigm Corporation. All other trademarks are the property of their respective owners.
Using Licensed ReagentsWe recommend that you purchase licensed reagents only from authorized sources. If you have any questions regarding whether you have a license to use particular reagents, you should contact the appropriate licensor and obtain clarification and their permission if necessary.
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