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Experimental Method

Using the C1™ Single-Cell Auto Prep System to Capture

Cells from Cell Culture and Perform Preamplification of MicroRNA Species with TaqMan® AssaysPN 100-6667 B1

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Fluidigm Experimental Method

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Disclaimer

This Experimental Method is provided “as is.” NO WARRANTIES ARE PROVIDED, EXPRESSED OR IMPLIED.ALL WARRANTIES, INCLUDING THE IMPLIED WARRANTIES OF FITNESS FOR PURPOSE, MERCHANTABILITY,AND NON-INFRINGMENT ARE EXPRESSLY DISCLAIMED. Every effort has been made to avoid errors in thetext, diagrams, illustrations, figures, and screen captures. However, Fluidigm assumes no responsibilityfor any errors that may appear in this publication. It is Fluidigm's policy to improve products as newtechniques and components become available. Therefore, Fluidigm reserves the right to change spec-ifications at any time. Information in this Experimental Method is subject to change without notice.Fluidigm assumes no responsibility for any errors or omissions. In no event shall Fluidigm be liable forany damages in connection with or arising from the use of this Experimental Method.

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Experimental Method Fluidigm

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Overview of Experimental Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Recommended Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9IFC Types and Related Scripts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Reagent Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Running a C1™ IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Using the Chip Map Loading Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Priming the IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Preparing the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Loading the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21(Optional) Starting the Tube Controls: Lysis and Reverse Transcription . . . . . . . . . . . . . . . . . . 22Imaging the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Running Lysis, Reverse Transcription, and PreAmp on the C1™ System . . . . . . . . . . . . . . . . . . . 22(Optional) Continuing the Tube Control: PreAmp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Harvesting the Amplified Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Running the Reaction Products on a 96.96 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . 28Appendix 1: Running the Tube Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Appendix 2: IFC Pipetting Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

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Introduction

This protocol allows the user to capture cells, perform specific reverse transcription of microRNA, and perform targeted preamplification of cDNA using the Fluidigm C1

™ Single-Cell Auto Prep System and C1

™ Single-Cell Auto Prep Integrated Fluidic Circuits (IFCs). The protocol explains all steps, including: capturing cells, staining for viability, imaging cells, lysing cells, performing reverse transcription and preamplification, and finally, harvesting the amplified products. It describes the procedure to evaluate the RNA content of cells and to harvest the products generated from a C1

™ Single-Cell Auto Prep IFC. Gene expression analysis of preamplified amplicons is then performed with 48.48 or 96.96 Dynamic Array™ IFCs using the BioMark™ System or BioMark™ HD System.

During the reverse transcription step, microRNAs are reverse-transcribed to cDNA using stem loop RT primers (from the Megaplex™ Primer Pools, Life Technologies) that are specific for mature microRNA species, and reagents from the TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies). In the preamplification step, PCR products are uniformly amplified from cDNA templates using the Megaplex™ PreAmp Primers (see reference 1 in “References” on page 6) and Single-Cell PreAmp Mix from the Ambion® Single Cell-to-CT™ Kit (Life Technologies). Preamplification enriches samples for loci of interest, maintains relative abundance between loci, and permits subsequent quantitative Cq information to be derived (to see figures of the reverse transcription and preamplification process, see reference 1 in “References” on page 6). Quantitative PCR is then performed on the BioMark™ System. For more information on preamplification, see Devonshire et al. BMC Genomics 12 (2011): 118.

The optional live/dead cell staining step uses the LIVE/DEAD® Viability/Cytotoxicity Kit (see reference 2 in “References” on page 6), 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 cells only if the integrity of the cell membrane has been lost.

Revision History

Revision Date Description of change

B1 23 December 2013

• Replaced “chip” with “IFC” where appropriate.• Updated descriptions of the STA scripts used for

microRNA Seq (see “IFC Types and Related Scripts” on page 10).

• Reformatted the tube control tables for clarity (see “Appendix 1: Running the Tube Controls” on page 30).

A1 24 July 2013 New document on use of the new C1™ IFC for mRNA

seq (5-10 μm) with small cells.

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Overview of Experimental Workflow

Figure 1 Overview of the cell capture procedure

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References

1 Megaplex™ Pools For microRNA Expression Analysis Protocol (Life Technologies, PN 4399721), http://tools.invitrogen.com/content/sfs/manuals/4399721c.pdf

2 LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells (Life Technologies, PN L-3224)

Fluidigm® Technical Note: Single-Cell MicroRNA Expression Profiling with the C1™ Single-

Cell Auto Prep System (PN 100-6859)

Fluidigm® BioMark™ HD Data Collection Software User Guide (PN 100-2451)

Fluidigm® Real-Time PCR Analysis Software User Guide (PN 68000088)

Fluidigm® C1™ Single-Cell Auto Prep System User Guide (PN 100-4977)

Fluidigm® Application Guidance: Single-Cell Analysis (PN 100-5066)

Minimum Specifications for Single-Cell Imaging (Fluidigm, PN 100-5004)

INCYTO C-Chip™ Disposable Hemocytometer, at:www.incyto.com/product/product02_detail.php

Required Reagents

Stored at -20 °C Ambion® Single Cell-to-CT™ Kit (Life Technologies, PN 4458237)

TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies, PN 4366596)

Megaplex™ Primer Pools, Human Pools Set v3.0 (Life Technologies, PN 4444750) or Rodent Pools Set v3.0 (Life Technologies, PN 4444766). Sets contain RT primer pools and PreAmp primer pools (10X concentration).

C1™ Single-Cell Auto Prep Module 2 Kit (Fluidigm, PN 100-5519)

• C1™ Loading Reagent (Fluidigm)

• C1™ DNA Dilution Reagent (Fluidigm)

• C1™ Harvest Reagent (Fluidigm)

• C1™ Preloading Reagent (Fluidigm)

(Optional) LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells (Life Technologies, PN L-3224)

TaqMan® MicroRNA Assays. Each TaqMan® MicroRNA Assay includes (Life Technologies, PN 4427975):

–One tube containing small RNA-specific RT primer at 5X concentration (not used in this protocol)–One tube at 20X concentration containing a mix of:

•Small RNA-specific forward PCR primer•Specific reverse PCR primer•Small RNA-specific TaqMan® MGB probe

TaqMan® MicroRNA Reverse Transcription Kit [Life Technologies, PN 4366596 (200 reactions); PN 4366597 (1000 reactions)]

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www.incyto.com/product/product02_detail.php

http://tools.invitrogen.com/content/sfs/manuals/4399721c.pdf


Stored at 4 ºC C1

™ Single-Cell Auto Prep Module 1 Kit (Fluidigm, PN 100-5518)

• C1™ Blocking Reagent (Fluidigm)

• C1™ Suspension Reagent (Fluidigm)

Thermo Scientific NP-40 Detergent Surfact-Amps Solution [10% (w/v) NP-40; Thermo Fisher Scientific, PN PI-28324]

Stored at room temperature C1

™ Cell Wash Buffer (Fluidigm) (also from C1™ Module 1 Kit)

Nuclease-free water (Teknova, PN W3330 or equivalent)

Magnesium Chloride (MgCl2) at 100 mM (Allele Biotechnology, PN ABP-PP-ERMGS01)

Low lint cloth (Fluidigm)

70% Ethanol in a squirt bottle

(Optional) INCYTO C-Chip™ Disposable Hemocytometer (Neubauer Improved, PN DHC-N01)

Figure 2 C1™ Single-Cell Auto Prep Module 1 Kit 100-5518

IMPORTANT: Store the C1™ Single-Cell Auto Prep Module 1 Kit at 4 ºC as soon as it is received.

C1™ Suspension

C1™ Blocking Reagent

C1™ Cell Wash

Reagent (50 μL)

100-5315

(180 μL) 100-5316

Buffer (25.9 mL) 100-5314

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=

Figure 3 C1™ Single-Cell Auto Prep Module 2 Kit 100-5519

Required Equipment

C1™ Single-Cell Auto Prep System

C1™ Single-Cell Auto Prep IFCs (1782x, 1783x, 1784x)

96-well PCR Plate (USA Scientific, TempPlate™ semi-skirted, PN 1402-9700)

2 centrifuges: 1 for Eppendorf tubes, 1 for 96-well plates

Vortexer

2-μL pipette

IMPORTANT: Store the C1™ Single-Cell Auto Prep Module 2 kit at -20 ºC as soon as it is received.

IMPORTANT: In order to measure any volume smaller than 2 μL, Fluidigm highly recommends using a pipette capable of measuring 0.1 μL to 2 μL or 0.1 μL to 3 μL in 0.002 μL increments, such as the Rainin Pipet-Lite XLS model, as required for the Lysis Final Mix and RT Final Mix.

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Recommended Equipment

2 biocontainment hoods to prevent DNA contamination of lab and samples

Imaging equipment compatible with C1™ Single-Cell Auto Prep Integrated Fluidic

Circuits (IFCs). For details, see Minimum Specifications for Single-Cell Imaging, PN 100-5004.

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IFC Types and Related Scripts

There are currently three IFCs (“chips”) for preamplification (PreAmp) protocols of small, medium, and large single cells:

Table 1 IFCs and related scripts

Cell Size/IFC Name and Part numbers (PNs)

Barcode (prefix) Scripts Description

Small (5-10 μm)

C1™ Single-

Cell Auto Prep IFC for PreAmp (5-10 μm) PN 100-5757

1784x

STA: Prime (1784x)

Priming the control line and cell capture channels of the 5-10 μm PreAmp IFC (1784x)

STA: Cell Load (1784x)

Cell loading and washing without staining for PreAmp of 5-10 μm diameter cells (1784x)

STA: Cell Load & Stain (1784x)

Cell loading, staining, and washing for PreAmp of 5-10 μm diameter cells (1784x)

STA: miRNA PreAmp (1784x)

Loading, thermal, and harvest protocol for single-cell lysis, reverse transcription, and cDNA preamplification for the 5-10 μm PreAmp IFC (1784x)

Medium (10-17 μm)

C1™ Single-


1782x

STA: Prime (1782x)



Cell loading and washing without staining for PreAmp of 5-10 μm diameter cells (1784x) for PreAmp of 10-17 μm diameter cells (1782x)



STA:miRNA PreAmp (1782x)


Large (17-25 μm)

C1™ Single-


1783x

STA: Prime (1783x)



Cell loading and washing without staining for PreAmp of 5-10 μm diameter cells (1784x) for PreAmp of 17-25 μm diameter cells (1783x)



STA:miRNA PreAmp (1783x)


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Safety

It is the individual’s responsibility to review all MSDSs for chemicals used in this procedure before running the test.As with all procedures, the following 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, material 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.

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 a 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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Reagent Mixes

Overview – Premixes to prepareThe following instructions prepare reagents sufficient for one IFC. All other reagents can be scaled up if running multiple IFCs simultaneously:

“Reagent Retrieval” on page 13

“Lysis Final Mix” on page 15

“Reverse Transcription (RT) Final Mix” on page 15

“PreAmp Final Mix” on page 16

“(Optional) Preparing LIVE/DEAD Cell Staining Solution” on page 17

NOTE: Allow C1™ Cell Wash Buffer, C1

™ DNA Dilution Reagent, and C1™ Harvest Reagent (Fluidigm

Corporation) to equilibrate to room temperature prior to use.

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Reagent Retrieval

Required Reagents Preparation Kit Name

Lysis Final Mix

Single-Cell Lysis Solution Remove from -20 ºC, thaw, and keep on ice Ambion® Single Cell-to-CT Kit (Life Technologies)

Thermo Scientific NP-40 Detergent Surfact-Amps Solution (10% NP-40)

Remove from 4 ºC and keep on ice (Thermo Fisher Scientific)

Megaplex™ RT Primer Pool

Remove from -20 ºC, thaw, and keep on ice Megaplex™ Primer Pools, Human/rodent Pools Set v3.0 (Life Technologies)

C1™ Loading

ReagentRemove from -20 ºC and thaw to room temperature

Fluidigm C1™ Module 2 Kit

RT Final Mix

Stop Solution Remove from -20 ºC, thaw, and keep on ice Ambion® Single Cell-to-CT Kit (Life Technologies)

10X RT Buffer Remove from -20 ºC, thaw, and keep on ice TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies)

MultiScribe™ RT Remove from -20 ºC and keep on ice TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies)

dNTP mix Remove from -20 ºC, thaw, and keep on ice TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies)

Magnesium Chloride (100 mM)

Keep at room temperature (Allele Biotech)

PreAmp Final Mix

Single-Cell PreAmp Mix Remove from -20 ºC, thaw, and keep on ice Ambion® Single Cell-to-CT Kit (Life Technologies)

Megaplex™ PreAmp Primer Pool

Remove from -20 ºC, thaw, and keep on ice Megaplex™ Primer Pools, Human/rodent Pools Set v3.0 (Life Technologies)

Nuclease-free water Keep at room temperature (Teknova)© Fluidig

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Table 2 Reagent supplies

Optional LIVE/DEAD Cell Staining

C1™ Cell Wash Buffer Remove from 4 ºC Fluidigm C1

™ Module 1 Kit

Ethidium homodimer-1 Remove from -20 ºC and keep in the dark as much as possible

LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies)

Calcein AM Remove from -20 ºC and keep in the dark as much as possible

LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies)

Priming

C1™ Blocking

ReagentRemove from 4 ºC and thaw to room temperature


C1™ Preloading

ReagentRemove from-20 ºC and thaw to room temperature


Cell Loading

C1™ Suspension

ReagentRemove from 4 ºC and keep on ice Fluidigm C1

™ Module 1 Kit

Required Reagents Preparation Kit Name

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Lysis Final Mix

1 After thawing the Single-Cell Lysis Solution from the Single Cell-to-CT Kit, vortex it for 3 seconds and spin briefly to collect contents.

2 Thaw the Megaplex™ RT primer pool on ice and mix by inverting six times. Spin briefly.3 In a DNA-free hood, prepare 12.0 μL of Lysis Final Mix by combining the following components in

this order:

Table 3 Lysis Final Mix

4 Vortex briefly then spin briefly to collect contents. Keep on ice until use.

Reverse Transcription (RT) Final Mix1 Thaw the Stop Solution from the Ambion Single Cell-to-CT Kit (Life Technologies), the 10X RT

buffer, and the dNTP mix from the TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies).

2 Vortex each reagent in the previous step for 3 seconds and spin briefly to collect contents. 3 Keep Multiscribe™ RT on ice. Flick the tube of enzyme to mix. Do not vortex the enzyme.

IMPORTANT: In order to measure any volume smaller than 2 μL, Fluidigm highly recommends using a pipette capable of measuring 0.1 μL to 2 μL or 0.1 μL to 3 μL in 0.002 μL increments, such as the Rainin Pipet-Lite XLS model, as required for the Lysis Final Mix and RT final mix.

Components Volume (μL)

Single-Cell Lysis Solution (Life Technologies) 8.50

Thermo Scientific NP-40 Detergent Surfact-Amps Solution 0.16

C1™ Loading Reagent 0.60

Megaplex™ RT pool (10X) 2.74

Total 12.00

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4 In a DNA-free hood, prepare 12 μL of RT (Reverse Transcription) Final Mix by combining the reagents in this order:

Table 4 RT Final Mix

5 Vortex the RT Final Mix briefly and spin briefly to collect contents. Keep on ice until use.

PreAmp Final Mix 1 Thaw the Megaplex™ PreAmp primers (Life Technologies) on ice and mix by inverting six times.

Spin briefly.2 In a DNA-free hood, prepare 60 μL of PreAmp Final Mix by combining reagents in this order:

Table 5 PreAmp Final Mix

3 Vortex the PreAmp Final Mix briefly and spin briefly to collect contents. Keep on ice until use.

IMPORTANT: In order to measure any volume smaller than 2 μL, Fluidigm highly recommends using a pipette capable of measuring 0.1 μL to 2 μL or 0.1 μL to 3 μL in 0.002 μL increments, such as the Rainin Pipet-Lite XLS model, as required for the Lysis Final Mix and RT final mix.


Stop solution (Life Technologies) 1.940

10X RT buffer (Life Technologies) 2.740

Multiscribe™ RT (Life Technologies) 5.490

dNTP mix 0.548

Magnesium Chloride (100mM) 0.686

C1™ Loading Reagent (Fluidigm) 0.600

Total 12.004


Single-Cell PreAmp Mix (Life Technologies) 13.0

Megaplex™ PreAmp pool (10X) 6.5

C1™ Loading Reagent (Fluidigm) 3.0

DNA-free Water 37.5

Total 60.0

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(Optional) Preparing LIVE/DEAD Cell Staining Solution

1 Vortex the dyes well before pipetting. 2 Prepare the LIVE/DEAD stain by combining the reagents in this order:

Table 6 Staining solution

3 Vortex the C1™ LIVE/DEAD staining solution well before pipetting onto the IFC.

Running a C1™

IFC

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 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 before pipetting into IFC.

NOTE: The concentration of Calcein AM may require optimization for some small cells (5–10 μm).


C1™ Cell Wash Buffer (Fluidigm)

(30 mL bottle)1250.0

Ethidium homodimer-1 (LIVE/DEAD kit, Life Technologies/Molecular Probes)

2.5

Calcein AM (LIVE/DEAD kit, Life Technologies/Molecular Probes)

0.625

Total 1253.125

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.

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Using 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 4 Chip Map Loading Plate

2 Place the C1™ Single-Cell Auto Prep IFC onto the Chip Map Loading Plate. For more details on

IFC loading, see “Appendix 2: IFC Pipetting Map” on page 33.3 Pipet the reagents (see “Priming the IFC” on page 19).

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Priming the IFC

To view the IFC pipetting map, see “Appendix 2: IFC Pipetting Map” on page 33

Figure 5 C1™ IFC priming pipetting map

1 Add 200 μL of C1™ Harvest Reagent from the 4-mL bottle into the accumulators marked with red

circles in Figure 5 using a pipette tip. 2 Pipet 20 μL of C1

™ Harvest Reagent into the wells marked with red squares on each side of the accumulators (36 total wells).

3 Pipet 20 μL of C1™ Harvest Reagent into the two wells on each side of the IFC in the middle of

the outside columns of wells 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 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™ Cell Wash Buffer from the 30-mL bottle into inlets 5 and 6 marked with dark

gray dots.7 Place the IFC into the C1

™ Single-Cell Auto Prep System then run the STA: Prime (1782x/1783x/1784x) script (see “IFC Types and Related Scripts” on page 10). When the Prime script has finished, press EJECT to remove the primed IFC from the instrument. This script takes approximately 10 minutes.

NOTE: After priming the IFC, you have 1 h to load the IFC with the C1™ Single-Cell Auto Prep

System.

#2 #5 #6

C1 Harvest Reagent, 20 μL

C1 Preloading Reagent, 20 μL

C1 Blocking Reagent, 15 μL

C1 Cell Wash Buffer, 20 μL


Key

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Preparing the Cells

1 Prepare a cell suspension of a concentration of 166-250 K/mL in native medium prior to mixing with C1

™ 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 IFC and the tube controls.

2 Prepare the cell mix by combining cells with C1™Cell Suspension Reagent at a ratio of 3:2.

For example:

Table 7 Cell mix

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/product/product02_detail.php for instructions for use.

CAUTION! Vortex the C1™ Suspension Reagent thoroughly prior to use. If C1

™ Suspension Reagent contains particulates, ensure they are properly removed by vortexing. DO NOT vortex the cells.


Cells 166-250K/mL 60

C1™ Cell Suspension Reagent (Fluidigm) 40

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

™ Cell Suspension Reagent of 3:2.

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Loading the Cells

Figure 6 C1™ IFC loading pipetting map

1 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.2 Remove blocking solutions from the cell inlet and outlet marked with teal and white dots in

Figure 6.3 Pipet 5–20 μL of Cell Mix into the cell inlet marked with the teal dot. Only 5 μL of Cell Mix 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

™ 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 STA: Cell Load (1782x/

1783x/1784x) or STA: Cell Load & Stain (1782x/1783x/1784x) script (see “IFC Types and Related Scripts” on page 10). When the script has finished, press EJECT to remove the IFC from the C1

™ Single-Cell Auto Prep System.

NOTE: Staining small cells (5–10 μm) takes 30 minutes, and staining medium (10–17 μm) or large (17–25 μm) cells takes 60 minutes.

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(Optional) Starting the Tube Controls: Lysis and Reverse Transcription

If you choose to start tube controls, see “Appendix 1: Running the Tube Controls” on page 30 for instructions.

Imaging the Cells

Cells may be imaged on a microscope compatible with C1™ IFCs. Guidelines for selection of a

microscope are outlined in Minimum Specifications for Single-Cell Imaging, PN 100-5004. Contact Fluidigm Technical Support for this document or with any questions. Call 1-866-358-4354 (within U.S.) or 1-650-266-6100 (outside U.S.), or email [email protected].

Running Lysis, Reverse Transcription, and PreAmp on the C1™ System

Figure 7 C1™ IFC Lysis, RT, and PreAmp pipetting map

1 Pipet 180 μL of C1™ Harvest Reagent into the four reservoirs marked with large solid red

rectangles in Figure 7.2 Pipet 7 μL of Lysis Final Mix in well #3, marked with an orange dot.3 Pipet 7 μL of RT Final Mix in well #4, marked with a yellow dot.4 Pipet 24 μL of PreAmp Final Mix in wells #7 and #8, marked with blue dots.

#3

#7 #8

Key

C1 Harvest Reagent, 1 0 μL

Lysis Final Mix, 7 μL

PreAmp Final Mix, 24 μL

Outputs Outputs

#3 #4

RT Final Mix, 7 μL

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5 Place the IFC into the C1™ Single-Cell Auto Prep System, choose the Specific Target Amplification

(STA) script: STA: miRNA PreAmp (1782x/1783x/1784x) script, then touch START (see “IFC Types and Related Scripts” on page 10).

NOTE: The STA: miRNA PreAmp (1782x/1783x/1784x) script may be run overnight. Approximate run times are:

• Small-cell IFC: ~7 hours (6 hours for lysis, reverse transcription, and preamplification; and 1 hour for harvest)

• Medium- and large-cell IFCs: ~8 hours (6 hours for lysis, reverse transcription, and preamplification; and 2 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 can be programmed to complete next morning:

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The STA: miRNA PreAmp (1782x/1783x/1784x) script contains the following thermal cycling protocols:

Table 8 Thermal cycling protocols

(Optional) Continuing the Tube Control: PreAmp

If you are running tube controls, see “Appendix 1: Running the Tube Controls” on page 30 for instructions.

Reverse Transcription

Stage Temperature Time Cycles

Anneal 16 ºC 2 min

40Extend 42 ºC 1 min

Extend 50 ºC 1 sec

Hold (enzyme inactivation)

85 ºC 5 min 1

Hold 4 ºC Hold

Preamplification


Enzyme activation/RT Inactivation

95 ºC 10 min 1

Hold 55 ºC 2 min 1

Hold 72 ºC 2 min 1

Denature 95 ºC 15 sec18

Anneal/Extend 60 ºC 4 min


99.9 ºC 10 min 1

Hold 4 ºC Hold

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Harvesting the Amplified Products

1 When the STA: miRNA PreAmp script has finished press 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 12.5 µL of C1™ DNA Dilution Reagent into each well of the DILUTED HARVEST PLATE.

5 Carefully pull the tape covering the harvesting inlets of the IFC using the plastic removal tool.

Figure 8 Tape removal

6 Using an 8-channel pipette, pipet the harvested amplicons from the inlets according to Figure 9 and Table 9 on page 26 and place in the DILUTED HARVEST PLATE:

Figure 9 Pipet map of reaction products on the C1™ IFC

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.

1 2 37 8 9 131415

4 5 610 11 1216 17 18131415 16 17 18

19 20 2125 26 2731 32 33

22 23 2428 29 3034 35 36

37 38 3943 44 45

40 41 4246 47 4843 44 45

49 50 51

46 47 4852 53 54

55 56 5761 62 6367 68 69

58 59 6064 65 6670 71 72

73 74 75 76 77 7873 74 7579 80 8185 86 87

76 77 7882 83 8488 89 90

91 92 93 94 95 96

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Table 9 Harvest amplicon dilution

7 Pipet the entire volume of C1™ harvest amplicons out of the left-side wells of the C1

™ IFC into the 12.5 μL of C1

™ DNA Dilution Reagent in each well of the DILUTED HARVEST PLATE:

Figure 10 First, second, and third pipetting steps

NOTE: Harvest volumes may vary. Set pipette to 3.5 μL to ensure entire volume is extracted.


C1™ DNA Dilution Reagent (Fluidigm)

(30 mL bottle)12.5

C1™ harvest amplicons ~3

Total ~15.5

IMPORTANT: These preamplified samples are now ready for analysis on the BioMark™ System or BioMark™ HD System, following the Fluidigm 96.96 Real-Time PCR Workflow Quick Reference (PN 68000130) with TaqMan® Gene Expression Master Mix (2X, Life Technologies, PN 4369016), instead of TaqMan® Universal PCR Master Mix (2X, PN 4304437).

For detailed instructions on pipetting the harvested aliquots to the “DILUTED HARVEST PLATE”, proceed to steps 7–10.

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

79 80 81

85 86 87

91 92 93

2

14

26

38

50

62

74

86

3

15

27

39

51

63

75

87

1 2 3 4 5 6 7 8 9 10 11 12

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8 Pipet the entire volume of C1™ harvest amplicons out of the right-side wells of the C1



Figure 11 Fourth, fifth, and sixth pipetting steps

9 Pipet the entire volume of C1™ harvest amplicons out of the left-side wells of the C1



Figure 12 Seventh, eighth, and ninth pipetting steps

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

16

28

40

52

64

76

88

5

17

23

29

41

53

65

77

89

6

18

30

42

54

66

78

90

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

33

45

57

69

81

93

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10 Pipet the entire volume of C1™ harvest amplicons out of the right-side wells of the C1



Figure 13 Tenth, eleventh, and twelfth pipetting steps

11 Run the diluted amplicons on the BioMark™ System or the BioMark™ HD System (see “Running the Reaction Products on a 96.96 Dynamic Array IFC”).

Running the Reaction Products on a 96.96 Dynamic Array IFC

1 Refer to Fluidigm 96.96 Real-Time PCR Workflow Quick Reference (PN 68000130, Rev. C).

2 In step 3 of the quick reference, use the TaqMan® Universal PCR Master Mix, No AmpErase® UNG (2X, Life Technologies, PN 4324018).

IMPORTANT: Always handle the reaction products from the C1™ IFC in the post-PCR lab.

NOTE: Follow the Fluidigm 96.96 Real-Time PCR Workflow Quick Reference, but perform steps 2–3 in this section, which are different from the specified steps in the quick reference.

IMPORTANT: Do not use the TaqMan® Universal PCR Master Mix (2X, Life Technologies, PN 4304437).

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

16

28

40

52

64

76

88

11

23

35

47

59

71

83

95

12

24

36

48

60

72

84

96

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3 In step 9 of the quick reference, do not run the suggested thermal cycling protocol. Program the following thermal cycling protocol instead, and save the protocol under the filename “GE 96x96 miRNA Standard v1”:

Table 10 BioMark™ System thermal cycling protocol

Figure 14 The BioMark™ thermal cycling protocol as it appears on the display

BioMark™ Real Time PCR Protocol (2 ºC/sec ramps)


Hold (thermal mix) 50 ºC 2 min 1



Hold (hot start) 95 ºC 10 min 1


Anneal 60 ºC 60 sec

NOTE: To change a thermal cycling protocol on the BioMark™ or BioMark™ HD System, refer to “The Protocol Editor” in the Fluidigm BioMark™ HD Data Collection Software User Guide (PN 100-2451).

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Appendix 1: Running the Tube Controls

Washing the Cells 1 Pellet remaining cells (1-mL volume is easiest). Speeds and durations may vary. We suggest

spinning cells at 300 x g for 5 minutes.2 Remove buffer from pellet by gently pipetting out the supernatant media without disturbing

the cell pellet.3 Resuspend cells in 1 mL of C1

™ Cell Wash buffer by pipetting up and down at least 5 times. This is wash 1.

4 Pellet cells again and remove supernatant.5 Wash a second time by resuspending in 1 mL of C1

™ Cell Wash buffer and pipet up and down 5 times.

6 Pellet cells a third time and remove supernatant.7 Resuspend cells in C1

™ Cell Wash Buffer approximately 90% of original volume, to keep original concentration, assuming a 10% loss.

8 Prepare two tube controls in new tubes:

Table 11 Tube controls without RT Final Mix9 Incubate the tube controls at room temperature for 5 minutes.10 Add to each tube control:

Table 12 Tube controls with all reagents11 Vortex briefly and spin to collect contents.12 Allow RT tubes to sit on ice (4 oC) for 5 minutes before transferring them to a thermal cycler.

Components Tube 1: Positive Control(μL)

Tube 2: NTC (no template control; μL)

Washed cells 1.0 —

C1™ Cell Wash Buffer — 1.0

Lysis Final Mix 2.0 2.0

Total 3.00 3.00

Components Tube 1: Positive Control(μL)

Tube 2: NTC (μL)

Incubated tube control (from previous step) 3.0 3.0

RT Final Mix 2.0 2.0

Total 5.00 5.00

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13 In a thermal cycler, run:

Table 13 Thermal cycling protocol14 After thermal cycling, combine the following in two tubes of a new PCR strip (one tube for the

cell sample and one tube for the NTC):

Table 14 PreAmp reaction

15 In a thermal cycler, run:

Table 15 PreAmp thermal cycling protocol

Reverse Transcription


Anneal 16 ºC 2 min

40Extend 42 ºC 1 min

Extend 50 ºC 1 sec


85 ºC 5 min 1

Hold 4 ºC Hold

Components Volume

PreAmp Final Mix 3.33 μL

RT Reaction 0.35 μL

Total 3.68 μL

Preamplification


Enzyme activation/RT Inactivation

95 ºC 10 min 1

Hold 55 ºC 2 min 1

Hold 72 ºC 2 min 1


Anneal/Extend 60 ºC 4 min


99.9 ºC 10 min 1

Hold 4 ºC Hold

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Diluting Products1 Transfer prepared material to a Post-PCR lab.2 Briefly vortex the prepared products and spin to collect content.3 Combine the following reagents:

Table 16 Dilution of PreAmp products

4 Store the diluted PreAmp products at -20 °C until use.

Running the Reaction Products on a 96.96 Dynamic Array IFCSee “Running the Reaction Products on a 96.96 Dynamic Array IFC” on page 28.


C1™ DNA Dilution Reagent (Fluidigm) 98

PreAmp Product 2

Total 100

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Appendix 2: IFC Pipetting Map

Overview of IFC pipetting

Figure 15 IFC pipetting map

#1 #2 #3 #4

#5 #6 #7 #8

Outputs Outputs

Prime the Chip

C1 Preloading Reagent, 20 μL

C1 Blocking Reagent, 15 μL

C1 Cell Wash Buffer, 20 μL



Key Lyse, RT and PreAmp

C1 Harvest Reagent, 1 0 μL

Lysis Final Mix, 7 μL

RT Final Mix, 7 μL PreAmp

Final Mix, 24 μL

Load the Chip

LIVE/DEAD Staining Solution, 20 μL

Cell Mix, 5-20 μL

REMOVE C1 Blocking Reagent

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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

© 2013 Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, BioMark, C1, and Dynamic Array are trademarks or registered trademarks of Fluidigm Corporation. All other trademarks are the property of their respective owners. For Research Use Only. Not for use in diagnostic procedures.

NOTICE TO PURCHASER: Disclaimer of License for PreAmp MethodsNo right to perform Life Technologies Corporation’s patented pre-amplification methods is conveyed with the purchase of reagents from Fluidigm Corporation. A license to perform such pre-amplification methods can be obtained (i) with purchase of a C1 Single-Cell Auto Prep IFC, Dynamic Array IFC, or Digital Array IFC from Fluidigm Corporation or (ii) by a separate license from Life Technologies Corporation. No right to resell the products and no other rights (such as real-time PCR methods, apparatus, reagents or software to perform digital PCR methods) are conveyed by Life Technologies Corporation expressly, by implication, or by estoppel. For information on obtaining additional rights, contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

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