C1 Script Builder USER GUIDE

Transcription

1 PN D1 C1 Script Builder USER GUIDE

2 For Research Use Only. Not for use in diagnostic procedures. Information in this publication is subject to change without notice. It is Fluidigm policy to improve products as new techniques and components become available. Therefore, Fluidigm reserves the right to change specifications at any time. Every effort has been made to avoid errors in the text, diagrams, illustrations, figures, and screen captures. However, Fluidigm assumes no responsibility for any errors or omissions. In no event shall Fluidigm be liable for any damages in connection with or arising from the use of this publication. Patent and Limited License Information Fluidigm products are covered by issued and pending patents in the United States and other countries. Patent and limited license information is available at fluidigm.com/legalnotices. Limited Use License to Perform Preamplification with Fluidigm IFCs A license to use Thermo Fisher Scientific's patented preamplification method workflows involving a Fluidigm integrated fluidic circuit (IFC) can be obtained (i) with purchase of a Fluidigm IFC from Fluidigm Corporation or (ii) by a separate license from Thermo Fisher Scientific. For licensing information, contact outlicensing@lifetech.com. Limited Digital PCR License A license to use Thermo Fisher Scientific's patented digital PCR method in all fields other than in the Sequencing Field, the Mass Spectrometry Field, and the Prenatal Field in workflows involving a Fluidigm IFC can be obtained (i) with purchase of a Fluidigm IFC from Fluidigm Corporation or (ii) by a separate license from Thermo Fisher Scientific. For licensing information, contact outlicensing@lifetech.com. Trademarks Fluidigm, the Fluidigm logo, C1, Open App, Script Builder, and Script Hub are trademarks or registered trademarks of Fluidigm Corporation in the United States and/or other countries. All other trademarks are the sole property of their respective owners. For EU's WEEE directive information, go to fluidigm.com/compliance Fluidigm Corporation. All rights reserved. 06/2016 For technical support visit fluidigm.com/support. North America Toll-free: in the US techsupport@fluidigm.com Europe techsupporteurope@fluidigm.com China (excluding Hong Kong) techsupportchina@fluidigm.com Japan techsupportjapan@fluidigm.com All other Asian countries techsupportasia@fluidigm.com Central and South America techsupportlatam@fluidigm.com 2

3 Contents About This Guide 5 Document Revision History 5 Chapter 1: Overview of C1 Script Builder 6 Get Started with C1 Script Builder 6 Menu Options 6 File Menu 7 Edit Menu 7 Protocol Menu 7 Publish Menu 8 Settings Menu 8 Help Menu 8 Tabs 9 Protocol Summary Tab 9 Reagent Assignment Tab 9 Protocol Recipe Tab 10 Script Review Tab 10 Chapter 2: Translate Your Chemistry into the C1 Open App IFC 11 C1 Loading Reagent 11 C1 Open App IFCs 11 IFC Definition 12 Reagents Supplied by Fluidigm 12 Chamber Volumes 13 Reagent Concentrations 14 Examples for Calculating Reagent Concentration 15 Preliminary Optimization in Tubes 16 Chapter 3: Create a Protocol 17 Default Templates 17 Default Scripts 18 C1 Script Builder Design Rules 19 C1 Script Builder Design Considerations 20 Create a New Protocol from a Default Template 21 Create a New Protocol from an Existing One 22 Create a New Protocol from Scratch 23 Open a Completed Protocol and Create a New Version 24 View a Previous Version of a Protocol 25 Delete a Version of a Protocol 25 Export a Protocol as a Template 26 Chapter 4: Customize a Protocol 27 Add, Remove, or Change the Order of Steps and Scripts 27 Add Steps and Scripts 27 Remove All Scripts from the Workspace 28 Edit or Remove a Script or Step Icon in the Modify Protocol Window 28 Change the Order of Steps 29 Change the Step Properties in the Reagent Assignment Tab 29 Change the Script Properties 31 Change the Incubation Parameters 32 The Incubation Pane 32 Set a Single Temperature and Duration for Incubation 33 Open the Thermal Protocol Editor 34 Modify a Thermal Protocol for Incubation 35 Change the Advanced Properties 36 Assign Chambers and Inlets to a Step 37 Clear Assignments to Inlets 38 Clear Assignments to Experiment Chambers 39 Reuse an Inlet 40 Script Steps 18 3

4 Contents Chapter 5: Manage Reagents 41 Protocol Recipe Tab 41 Reagent Mixes Pane 42 Reagent Mix Definition Pane 42 Modify an Assigned Reagent Mix 43 Assign a Different Reagent Mix 44 Define a New Reagent Mix 44 Add, Import, or Remove Reagent Components 45 Adjust Concentrations and Volume Values 46 Reagent Mix Library 47 Add a New Reagent Mix to the Reagent Library 48 Export the User Library 49 Import a User Library 50 Reagent List 50 Chapter 6: Generate and Run Scripts 51 Generate the Scripts and Protocol Worksheet 51 Running Custom Scripts 52 Run Scripts on the C1 System from the USB Drive 52 Install Protocols on the C1 System 53 Protocol Worksheet 54 Appendix B: Request and Add a New or Updated License File 60 Generate a C1 System Key 60 Request a License File 61 Add the License File into C1 Script Builder 62 Drag the License File into C1 Script Builder 62 Import the License File into C1 Script Builder 62 Appendix C: Create and Modify Thermal Cycling Protocols 63 Thermal Protocol Editor User Interface 63 Create a Thermal Cycling Protocol 64 Working with Segments and Slices 65 Change a Segment Name or Ramp Rate 65 Add a New Segment 66 Remove a Segment 66 Change Slice Properties 66 Add a Slice to a Segment (manual protocol only) 67 Remove a Slice from a Segment (manual protocol only) 67 Working with Ramp Rates 67 Change the Ramp Rate for a Protocol 67 Mix Ramp Rates in a Protocol 67 Chapter 7: Submit a Protocol for Publication on Script Hub 55 Publication Summary 55 Submit a Protocol for Sharing 56 Appendix A: Install C1 Script Builder 58 System Requirements 58 Install C1 Script Builder on Your Computer 58 Update the C1 Script Builder Software 59 Update Scripts to Use the New IFC Definition 59 4

5 About This Guide Document Revision History For detailed information about the software releases, see the C1 Script Builder Release Notes (PN ). Revision Date Description of change D1 20 June 2016 Updated for v2.1.1 of the C1 Script Builder software. Changed cell-capture volume from 400% to 300% to support redesigned medium-cell integrated fluidic circuits (IFCs) (page 20 and page 36). Updated IFC definition (page 12). Updated instructions for downloading and installing the software (Appendix A) and requesting a license file (Appendix B). Added information about the trial mode of the software (various pages throughout). Updated IFC images (various pages throughout). C1 19 May 2015 Updated for v of the C1 Script Builder software. Changed all instances of C1 Script Hub to Script Hub (various pages throughout). Replaced description with source in the Tested Cells tab (see page 56). Updated formatting. B1 3 February 2015 Added the Protocol Recipe tab. Added instructions for submitting a protocol for publication on C1 Script Hub. Updated formatting. A2 17 June 2014 New user guide for using the C1 Script Builder software 5

6 Chapter 1: Overview of C1 Script Builder You can use C1 Script Builder to develop new protocols for the C1 system or modify existing ones. With C1 Script Builder, you define the load, lysis, wash, and incubation (thermal cycling) parameters and reagents. You can then generate scripts for the C1 system. Get Started with C1 Script Builder The C1 Script Builder software can be downloaded from fluidigm.com/software. You can use C1 Script Builder in trial mode to create protocols and design custom scripts, but to generate and use the scripts on your C1 system you must purchase a license. For information about installing C1 Script Builder, see Appendix A on page 58. For information about licenses, see Appendix B on page 60. Start C1 Script Builder by double-clicking the C1 Script Builder icon ( desktop. ) on the Menu Options The C1 Script Builder start screen shows the File, Settings, and Help menus. The Edit, Protocol, and Publish menus appear when a protocol is open. 6

7 Chapter 1: Overview of C1 Script Builder Menu Options File Menu From the File menu you can: Create a new protocol from scratch (File > New). Create a new protocol from an existing one (File > New from existing). Open an existing protocol (File > Open) or select from a list of recently saved protocols (File > Recent). Close an open protocol (File > Close). Save an open protocol to a user-defined folder on the computer (File > Save). Save a copy of the protocol using a new filename and/or folder (File > Save As). The version history in the saved copy will be the same as the original protocol. To create a copy of a protocol that does not include the version history of the original protocol, use the New from existing command. Export a single version of the protocol as a template file (File > Export). Exit C1 Script Builder (File > Exit). Edit Menu After opening an existing protocol or creating a new protocol, use the Edit menu options to: Add or remove scripts and steps from the protocol or clear the entire protocol and start over. Clear the reagent inlet and chamber assignments (Edit > Clear Assignments). Check for errors and warnings in your protocol (Edit > Design Rule Check). Protocol Menu When you have completed your protocol and are ready to run the scripts on the C1 system, use the Protocol menu options to: Generate scripts and a PDF worksheet for the custom script (Protocol > Generate Scripts). Preview the worksheet (Scripts > Preview Protocol Worksheet). Review the Fluidigm-supplied reagent kits used in the protocol (Protocol > Reagent List). 7

8 Chapter 1: Overview of C1 Script Builder Menu Options Publish Menu If you are submitting your protocol for publication on Script Hub, use the Publish menu options to: Enter information describing your protocol (Publish > Publication Summary). Submit a script to Fluidigm for publication on Script Hub (Publish > Submit to Script Hub). Settings Menu From the Settings menu, you can: Import license files (Settings > License Management). Set up and manage a reagent mix library (Settings > Reagent Mix Library). Help Menu From the Help menu, you can: Access the user documentation on the Fluidigm website (Help > Download User Documentation). View the version information for C1 Script Builder (Help > About). 8

9 Chapter 1: Overview of C1 Script Builder Tabs Tabs After opening an existing protocol or creating a new protocol, use the tabs to modify script properties and view details. Protocol Summary Tab In the Protocol Summary tab, you can view and modify protocol properties, including name, description, IFC (integrated fluidic circuit) definition, version history, authors, institution, lab, and any special instructions. In this tab, you can select a version to view or edit or manage versions by creating new versions and deleting unwanted ones. Reagent Assignment Tab In the Reagent Assignment tab, you can assign reagents to inlets and chambers, and incubation parameters to steps and scripts. For more information, see Chapter 4: Customize a Protocol. 9

10 Chapter 1: Overview of C1 Script Builder Tabs Protocol Recipe Tab In the Protocol Recipe tab, you can review the reagents and reagent mixes used in each step in a script. In this tab, you can add or remove reagents in the script, calculate mix volume, and add reagents to the reagent library. For more information, see Chapter 5: Manage Reagents. Script Review Tab In the Script Review tab, you can view incubation parameters, reagents, and IFC inlet assignments for each script in your protocol. The Script Review tab contains three additional tabs: Script View tab. Click a script in the preview area to view incubation parameters and calculate a time estimate for a script, depending on the selected IFC barcode. Reagents tab. Click a script in the preview area to view reagents used. IFC Inlet Assignments tab. Click a script in the preview area to view a map of the integrated fluidic circuit (IFC) showing inlets assigned to steps in the script. 10

11 Chapter 2: Translate Your Chemistry into the C1 Open App IFC This chapter describes how to port your chemistry from tubes to the C1 Open App IFC (integrated fluidic circuit) when creating custom protocols in C1 Script Builder for use with a C1 system. C1 Loading Reagent All reagent mixes must include C1 Loading Reagent to ensure efficient loading into the reaction chambers of the C1 Open App IFC. C1 Loading Reagent (PN ) is supplied as a 20X stock in all C1 reagent kits. It should be added to all reagent mixes to a final concentration of 1X. C1 Open App IFCs To run custom C1 scripts, use the following C1 Open App IFCs: Part Number Description IFC Barcode C1 Open App IFC for small cells (5 10 μm) 1861x C1 Open App IFC for medium cells (10 17 μm) 1862x C1 Open App IFC for large cells (17 25 μm) 1863x The C1 Open App IFC is the only IFC that can be used to run custom C1 scripts. CAUTION The composition of the IFC includes materials that might be sensitive to harsh chemicals, especially when elevated temperatures are required. To avoid damaging IFC and instrument and possibly causing injury, do not load non-aqueous solutions or solutions that are strongly acidic (ph <2) or basic (ph >12). For cases where the use of acids or bases is necessary, check the box Require Neutralize Reagent in the Reagent Assignment tab. This enables removal of potentially damaging reagents from the IFC lines after their use, allowing safer fluid handling in the IFC and in its frame. IMPORTANT Avoid using hydrophobic small molecules (logd >3.5), since they might be partially absorbed by the polydimethylsiloxane (PDMS) matrix, which can cause the effective molecular concentration to be lowered. 11

12 Chapter 2: Translate Your Chemistry into the C1 Open App IFC IFC Definition IFC Definition The IFC definition determines which versions of Open App IFCs are supported by the C1 Script Builder software. The current IFC definition is assigned automatically to new protocols, new versions of existing protocols, and in-progress versions of existing protocols. To update scripts to use the new IFC definition in completed versions of protocols, see page 59. V01.1 is the second IFC definition version for Open App IFCs. Compatibility for small-cell and large-cell IFCs has not changed, but the redesigned medium-cell Open App IFCs are compatible only with V01.1. NOTE V01.1 is not compatible with the original design of the medium-cell Open App IFCs. Reagents Supplied by Fluidigm Custom scripts made with C1 Script Builder require the use of certain reagents supplied by Fluidigm for priming the IFC, as well as for loading cells and reagents. For custom scripts that represent incremental modifications of existing default templates and chemistry, use the applicable reagent kit for the default template you select. Prepare reagents according to instructions described in the corresponding protocol. For custom scripts that are not based on existing protocols, the required Fluidigm reagents should be obtained from the Open App Reagent Kit (PN ). Application Reagent Kit Part Number Instructions for Preparation Programmable DNA Seq Programmable mrna Seq Programmable STA C1 Reagent Kit for DNA Seq C1 Reagent Kit for mrna Seq C1 Reagent Kit for Preamp Custom Open App Reagent Kit Defined by user Open App Loading Reagent Using C1 to Generate Single-Cell Libraries for DNA Sequencing (PN ) Using C1 to Generate Single-Cell cdna Libraries for mrna Sequencing (PN ) Using C1 to Capture Cells from Cell Culture and Perform Preamplification Using Delta Gene Assays (PN ) For more information about the default templates, see page 17. For more information about using the C1 system, see the C1 System User Guide (PN ). 12

13 Chapter 2: Translate Your Chemistry into the C1 Open App IFC Chamber Volumes Chamber Volumes The volumes for the experimental chambers of the C1 Open App IFC are as follows: C (capture site), 4.5 nl E1 (chamber 1), 9 nl E2 (chamber 2), 9 nl E3 (chamber 3), 9 nl E4 (chamber 4), 135 nl E5 (chamber 5), 135 nl The reaction chambers of the C1 Open App IFC fill sequentially from chamber 1 to chamber 5 and include the capture sites. Once filled, a chamber is connected to all previous chambers; the total reaction volume is the sum of the volumes of all filled chambers. For example, when reagents are added to chamber 3, the total reaction volume is the sum of the volumes of the capture sites and chambers 1 to 3 (4.5 nl + 9 nl + 9 nl + 9 nl = 31.5 nl). 13

14 Chapter 2: Translate Your Chemistry into the C1 Open App IFC Reagent Concentrations Reagent Concentrations Because reagents added to reaction chambers are diluted by the total volume of the previous chambers, the reagents loaded onto the IFC must be loaded at higher initial concentrations to achieve the desired concentration of 1X in the reaction chambers. Table 1 provides the concentrations for reagents that must be loaded into the inlet of the IFC to achieve a 1X concentration when filling the various possible combinations of reaction chambers. Table 1. Concentrations for reagents using multiplication factors Chamber(s) Already Filled Going into Chamber(s) Added Reagents at C1 Loading Reagent Capture site 1 1.5X 1X Capture site X 1X Capture site X 1X Capture site X = 1X 1X Capture site X = 1X 1X Capture site X 1X Capture site X 1X Capture site X 1X Capture site X = 1X 1X Capture site X 1X Capture site X 1X Capture site X 1X Capture site X 1X Capture site X 1X Capture site X 1X 14

15 Chapter 2: Translate Your Chemistry into the C1 Open App IFC Reagent Concentrations Examples for Calculating Reagent Concentration The reagent mix calculator in C1 Script Builder can be used to calculate appropriate starting concentrations to achieve the desired final concentration in the Open App IFC. If desired, manual calculations can be performed as follows. Adding Reagents to Chamber 1 If you create a mix step that adds reagents to chamber 1, 9 nl of the reagents will be added to the chamber. Because the chambers fill sequentially and chamber 1 is contiguous with the capture site, a total volume of 13.5 nl (4.5 nl + 9 nl) is created. Therefore, the reagents must be loaded at 1.5X to achieve a final concentration of 1X in the combined chambers (13.5/9 = 1.5X). Adding Reagents to All Other Chambers This section uses adding reagents to chambers 1 and 2 as an example for adding reagents to all other chambers. If you create a mix step that adds reagents to chambers 1 and 2, 9 nl of the reagents will be added to both chambers. Because the chambers fill sequentially and chamber 1 and chamber 2 are contiguous with the capture site, a total volume of 22.5 nl (4.5 nl + 9 nl + 9 nl) is created. Therefore, the reagents must be loaded at 1.25X to achieve a final concentration of 1X in the combined chambers (22.5/18 = 1.25X). 15

16 Chapter 2: Translate Your Chemistry into the C1 Open App IFC Preliminary Optimization in Tubes Preliminary Optimization in Tubes IMPORTANT All reagent mixes must include C1 Loading Reagent to ensure efficient loading into the reaction chambers of the C1 Open App IFC. C1 Loading Reagent (PN ) is supplied as a 20X stock in all C1 reagent kits. It should be added to all reagent mixes to a final concentration of 1X. Test and optimize in tubes first. The concentration of each component in the reagents mix should be tested and optimized by running in tubes first before attempting reactions in the C1 Open App IFC. Tube reactions should include 1X C1 Loading Reagent. To test in tubes, multiply all volumes in the experimental chambers by 1,000. So, rather than starting with the 4.5 nl capture site, start with 4.5 μl. Because you are increasing the volume 1,000-fold, the input material should also increase 1,000-fold. Rather than starting with a single cell, use 1,000 cells in 4.5 μl cell wash buffer. To test with purified RNA or DNA, start with the approximate amount expected for 1,000 cells in 4.5 μl cell wash buffer. For RNA, this might be 10 ng of total RNA; for DNA this might be 6 ng of genomic DNA for human samples. Testing in tubes should follow the same sequential reagent additions and dilutions that occur on the IFC. The appropriate volume of reagents should be added to the entire reaction from the previous step. To simulate reagent additions to the first three chambers, 9 μl volumes can be added to the initial 4.5 μl. To simulate the larger 135 nl chambers (4 and 5), 135 μl of reagent can be added to the tube. Alternatively, 3.15 μl (10%) of the reaction volume representing the capture site and chambers 1 to 3 can be transferred to a new tube, and 100-fold volumes (13.5 μl each) can be used to simulate reagent additions to chambers 4 and 5. Vary the concentrations of critical components. To get the best results, each step in your protocol should be optimized. This is done by systematically varying the concentration of critical components and testing to see which combinations give the best result. The most comprehensive way to do this is by using design of experiments (DOE) matrices. Because there is carryover from reaction to reaction, you might find that concentrations of some components are lower or higher than what you might use if you are setting up single, stand-alone reactions. 16

17 Chapter 3: Create a Protocol A protocol consists of a set of scripts. Each script contains a series of individual steps. Script Step Protocol To create a protocol for use on the C1 system: Chapter 3 Chapter 4 Chapter 4 and Chapter 5 Chapter 6 Create a new protocol from a predefined template, from an existing protocol, or from scratch. OR Open an existing protocol and create a new version. Add, remove, and change the order of steps and scripts as necessary. Modify the protocol as necessary by: Changing incubation parameters Assigning reaction chambers Assigning Generate the scripts and the protocol worksheet. reagent inlets Defining reagent mixes Default Templates The templates in the Default Templates list in C1 Script Builder are based on the DNA Seq, mrna Seq, and STA applications released by Fluidigm. They are provided for your convenience so you can create new scripts by modifying reaction parameters in existing scripts. If you are implementing a new chemistry, these templates may not apply to your experiment. We have verified that these template scripts perform similarly to their non-script Builder counterparts, although the reagent volume, inlet assignment, and precise mechanics of these template scripts may differ slightly from standard scripts Fluidigm provides with the C1 system. 17

18 Chapter 3: Create a Protocol Default Scripts Default Scripts The scripts in the Default Scripts list contain the steps needed to perform a specific part of a protocol, including loading and staining the cells, preparing the sample, etc. You can use the following default scripts as a starting point for your custom scripts: Capture. Contains the capture step. New. Does not contain any steps. Harvest. Contains the harvest step. Single Script. Contains both a capture and a harvest script. Script Steps Each step in the Script Steps list is a specific operation within a script. These are the steps available for building custom C1 scripts: Flowthrough. Flows reagents through all the capture sites. In a flowthrough step, cells are still in fluidic communication. Use for staining, washing, dosing, etc. Mix. Adds reagents to defined reaction chambers. During a mix step, the cells are isolated, and chambers are mixed after loading. Use for lysis, incubations, and reactions (for example, reverse transcriptions or PCR), etc. Backload. Adds a specific reagent to each single cell. In the backload step, reagents are backloaded from the harvest well to the corresponding reaction chamber for the step. Use for barcodes, molecular tags, or any other reagent that should be specifically added to individual single-cell reactions. The capture and harvest steps are available only as part of the default scripts. Capture. Loads cells onto the C1 Open App IFC. This step is contained in the Capture and Single Script scripts. Harvest. Outputs the final reaction products to the harvest wells. This step is contained in the Harvest and Single Script scripts. Steps cannot be added after the harvest step. You can change the name and color for each step. See Edit or Remove a Script or Step Icon in the Modify Protocol Window on page 28 for more information. 18

19 Chapter 3: Create a Protocol C1 Script Builder Design Rules C1 Script Builder Design Rules C1 Script Builder validates scripts according to the following design rules: The maximum volume for an inlet is 27 μl. There cannot be more than one capture step in the protocol. All flowthrough steps must come before the mix and backload steps. All mix and backload steps must be in the same script. There cannot be more than one backload step in the protocol. There cannot be a total of more than five mix and backload steps in a protocol. Steps cannot be inserted between a neutralize step and its associated mix step. There cannot be more than one harvest step in the protocol. If a harvest step is used, it must be the last step in the protocol. When reusing reagent inlets, a reagent can be used for multiple steps in the same script as long as total calculated usage does not exceed 27 μl. When reusing reagent inlets, neutralized reagent inlets and neutralizing reagent inlets cannot be reused. When you have multiple flowthrough steps, the total volume of the cell capture step and all flowthrough steps in a single script cannot exceed 27 μl. 19

20 Chapter 3: Create a Protocol C1 Script Builder Design Considerations C1 Script Builder Design Considerations When designing a script, take the following into consideration: Active reagent mixing is supported for reactions ending in chambers 2, 3, and 5. The default mixing time is for loading reagents at room temperature or higher. If you load a reagent at a lower temperature, we recommend that you extend the mixing time. For reagents loading at 4 C, we suggest doubling the default mixing time. The maximum allowed additional mixing time is 1,500 seconds. For reagents with slow reaction kinetics, you can select Continue mixing during incubation to enable mixing during incubation or thermal cycling can improve results. IMPORTANT Continuous mixing is not recommended for long incubation steps such as PCR. Continuous mixing over extended time could reduce the lifetime of your C1 system. You can specify additional load time if the reagent used has high viscosity. The maximum additional load time is 1,200 seconds. The range of flowthrough volume is 50% to 300%, relative to the recommended volume/percentage. C1 Script Builder will automatically calculate the new volume required to dispense into the reagent inlet when flowthrough volume is changed. The range of cell capture volume is 50% to 300% relative to the recommended volume/percentage. C1 Script Builder will automatically calculate the additional volume required to dispense into the cell inlet. If a mix step uses both chambers 4 and 5, it requires two reagent inlets. A backload step can load into: Chamber 1 Chambers 1, 2, 3 Chambers 2, 3 Chamber 3 Chambers 4, 5 Chamber 5 20

21 Chapter 3: Create a Protocol Create a New Protocol from a Default Template Create a New Protocol from a Default Template 1 Select File > New. 2 In the New Protocol window: a b Enter a name for your protocol in the Name field. (Optional) Enter a description of your protocol. 3 Insert a template by either: Selecting a default template and dragging it into the workspace Clicking Import and selecting a saved custom template 4 If desired, add, remove, or change the order of the steps (see Add, Remove, or Change the Order of Steps and Scripts on page 27). Click OK. 5 In the Protocol Summary tab, enter additional project information, including a short description, special instructions, and the name of the author(s), institution, and lab. 6 Modify the parameters for the steps (see Chapter 4: Customize a Protocol). 21

22 Chapter 3: Create a Protocol Create a New Protocol from an Existing One Create a New Protocol from an Existing One You can create a new protocol by copying an existing one. 1 Open the protocol you want to copy. 2 Select File > New from existing. 3 In the New Protocol window: a b c d Select the version of the protocol you want to copy. The version you select will display in the preview area. Enter a name for your protocol in the Name field. (Optional) Enter a description of your protocol. Click OK. 4 If desired, add, remove, or change the order of the steps (see Add, Remove, or Change the Order of Steps and Scripts on page 27). Click OK. 5 In the Protocol Summary tab, enter additional project information, including a short description, special instructions, and the name of the author(s), institution, and lab. 6 Modify the parameters for the steps (see Chapter 4: Customize a Protocol). 22

23 Chapter 3: Create a Protocol Create a New Protocol from Scratch Create a New Protocol from Scratch 1 Select File > New. 2 In the New Protocol window: a b Enter a name for your protocol in the Name field. (Optional) Enter a description of your protocol. 3 Select a default script and drag it into the workspace. 4 Add, remove, or change the order of the steps (Add, Remove, or Change the Order of Steps and Scripts on page 27). Click OK. 5 In the Protocol Summary tab, enter additional project information, including a short description, special instructions, and the name of the author(s), institution, and lab. 6 Modify the parameters for the steps (see Chapter 4: Customize a Protocol). 23

24 Chapter 3: Create a Protocol Open a Completed Protocol and Create a New Version Open a Completed Protocol and Create a New Version To make modifications after you generate your scripts and save the completed version of the protocol, you need to create a new version. NOTE The current IFC definition is assigned automatically to new protocols, new versions of protocols, and any versions of protocols that are in progress. Open a completed protocol by either: Selecting File > Open Selecting File > Recent. 1 In the History pane, click New version. 2 By default, the new version will use the current version as a starting point. If you want to use a version other than the current one as the starting point for the new version, select it from the dropdown list. 3 Click OK. If desired, you can change the name and description of the new version in the Protocol Summary tab and enter additional project information, including a short description, special instructions, and the name of the author(s), institution, and lab. 24

25 Chapter 3: Create a Protocol View a Previous Version of a Protocol View a Previous Version of a Protocol In the History pane, click the version number you want to view. If desired, you can change the description of a completed version. Delete a Version of a Protocol 1 In the History pane, click Delete version. 2 Select the version you want to remove in the Delete Version dropdown list. Click OK. NOTE Subsequent versions will not be renumbered. 3 Click OK. 25

26 Chapter 3: Create a Protocol Export a Protocol as a Template Export a Protocol as a Template You can export a version of a protocol as a template to import into a new protocol. 1 Open the protocol you want to use as a template. 2 Select File > Export. 3 Select the version of the protocol to export (if not using the latest version). 4 Enter a name and a description for the template. 5 Click OK. 26

27 Chapter 4: Customize a Protocol You can customize protocols by: Adding, removing, or changing the order of steps and scripts. Changing incubation parameters. Assigning reaction chambers and reagent inlets. Add, Remove, or Change the Order of Steps and Scripts When you create a new protocol, you can change the steps and scripts while in the New Protocol window. After you have closed the New Protocol window, you can still add, remove, or change the order of steps and scripts by selecting Edit > Modify Protocol. After the steps and scripts are arranged as desired, click OK. Add Steps and Scripts Add steps and scripts by dragging script and step icons from the list boxes into the template. 27

28 Chapter 4: Customize a Protocol Add, Remove, or Change the Order of Steps and Scripts C1 Script Builder will validate the scripts before leaving the Modify Protocol window and alert you of any errors in your protocol. For information about the design rules, see C1 Script Builder Design Rules on page 19. Remove All Scripts from the Workspace Click Clear to remove all the scripts in the workspace and start over. Edit or Remove a Script or Step Icon in the Modify Protocol Window 1 Click the script title or the step icon. Click title to edit or remove a script Click icon to edit or remove a step 2 In the Edit Protocol Step dialog box you can: Enter a new name for the script or step. (Steps only) Select a new color for the icon. Remove the script or step by selecting the checkbox. You can also change the name and color of steps and scripts in the Reagent Assignment tab. 28

29 Chapter 4: Customize a Protocol Change the Step Properties in the Reagent Assignment Tab Change the Order of Steps To change the order of steps in a script, you can: Drag steps to a new position in the same script. Copy a step by dragging it into a different script. Change the Step Properties in the Reagent Assignment Tab The Properties pane is in the Reagent Assignment tab. 1 Click a step to select it. 29

30 Chapter 4: Customize a Protocol Change the Step Properties in the Reagent Assignment Tab 2 In the Properties pane, enter the desired step name and assign a display color for the selected step. 3 In the Reagent Mix field, enter the name of the reagent mix used in the step or click and select a reagent mix from the library. IMPORTANT If you are planning to share your protocol on Script Hub, you must define all reagent mixes used in all the scripts. 4 For any mix steps that require neutralization, select the Require Neutralize Reagent Mix checkbox. This automatically inserts a neutralize step after that mix step. The IFC inlet assigned to the neutralizing reagent is predefined by the system. 30

31 Chapter 4: Customize a Protocol Change the Script Properties Change the Script Properties 1 Select the Reagent Assignment tab to access the Properties pane. 2 Click in a script to view the properties for the script. 3 If desired, in the Script Properties pane you can change the name of the script, enter a description, and enter any special instructions. 31

32 Chapter 4: Customize a Protocol Change the Incubation Parameters Change the Incubation Parameters The Incubation Pane The Incubation pane is in the Reagent Assignment tab. You can select from the following incubation parameter types: Single Step. Specify a single temperature and duration for incubation. Room Temperature. Specify only the duration for incubation. Protocol. Modify existing multiple-cycle thermal protocols or create new ones. None. No incubation needed. For mix steps, reagents are actively mixed after the loading of the new reagent is complete for additions to chambers 2, 3, and 5 before the incubation begins. Selecting the Continuous mixing during incubation checkbox will cause active mixing to continue during the incubation to improve results when using reagents that have slow reaction kinetics. IMPORTANT Continuous mixing is not recommended for long incubation steps such as PCR. Continuous mixing over extended time could reduce the lifetime of your C1 system. 32

33 Chapter 4: Customize a Protocol Change the Incubation Parameters Set a Single Temperature and Duration for Incubation 1 In the Reagent Assignment tab, click a step to select it. 2 In the Incubation pane, click the Type dropdown menu. Select either Single Step or Room Temperature. OR 3 (Single Step only) In the Temperature field, enter the desired temperature, up to 99 C. 4 In the Duration field, enter the desired number of seconds, up to 86,400 (24 hours). 5 Confirm your changes by clicking the step again and reviewing the Incubation pane. 33

34 Chapter 4: Customize a Protocol Open the Thermal Protocol Editor Open the Thermal Protocol Editor For more information about using the Thermal Protocol Editor, see Appendix C on page In the Reagent Assignment tab, click a step to select it. 2 In the Thermal Protocol field, click to open the Thermal Protocol Editor. NOTE If the Thermal Protocol field is not active, click the Type drop-down menu and select Protocol. 34

35 Chapter 4: Customize a Protocol Open the Thermal Protocol Editor Modify a Thermal Protocol for Incubation In the Thermal Protocol Editor, you can: Click the icons to edit, insert, or remove slices and segments. Segment Slice Double-click a slice to change its name, temperature, duration, or ramp rate. Double-click a segment to change its name, number of repeats, or ramp rate. 35

36 Chapter 4: Customize a Protocol Change the Advanced Properties Change the Advanced Properties The Advanced Properties pane is in the Reagent Assignment tab. Each type of step has a set of parameters that you can adjust. For a capture step, you can change the: Capture volume percentage. Increases or decreases the volume of cell suspension loaded into the capture sites. You can adjust the volume percentage from 50% up to 300%. Default setting is 100% (6 μl) Load temperature. Sets the temperature at which reagents load, ranging from 4 to 40 C. Default temperature is 25 C. For flowthrough steps, you can change the: Flowthrough volume percentage. Increases or decreases the operation time depending on the amount of reagent flowing over the capture sites. You can adjust the volume percentage from 50% up to 300%. Default setting is 100%. Flowthrough temperature. Sets the temperature at which reagents load, ranging from 4 to 40 C. Default temperature is 25 C. For mix steps, you can change the: Load temperature. Sets the temperature at which the reagents load, ranging from 4 to 40 C. Default temperature is 25 C. Additional load time. Increases the time allowed for loading the reagent up to an additional 1,200 seconds. Original load time is displayed in the Load time field in seconds. 36

37 Chapter 4: Customize a Protocol Assign Chambers and Inlets to a Step For mix steps including chambers 2, 3, and 5, you can set the: Additional mix time. Increases the time allowed for mixing the reagent after loading up to an additional 1,500 seconds. The original mix time is displayed in the Mix time field. Mixing temperature. Sets the incubation temperature during the mix step, ranging from 4 to 99 C. Default temperature is 25 C. For a harvest step, you can change the: Harvest temperature. Reduces the temperature at which amplified products are harvested, ranging from 10 to 25 C. Default temperature is 25 C. Pre-harvest temperature. Sets the temperature at which amplified products are held ranging from 10 to 25 C, if amplified products are not being harvested immediately after the run. Default temperature is 10 C. Assign Chambers and Inlets to a Step Each step requires at least one reagent inlet to be assigned to it. In addition, each mix and backload step requires at least one experimental chamber be assigned to it. 1 In the Reagent Assignment tab, click an unassigned step (without a check mark). 2 Select the experiment chamber(s) needed for the step. NOTE If a step does not need chambers assigned, the chamber graphics are grayed out. Step that needs chamber assigned Click to assign 37

38 Chapter 4: Customize a Protocol Clear Assignments to Inlets 3 Click an inlet on the IFC Inlets diagram and assign the reagent inlet. The diagram shows the number of inlets that you need to assign for the step. 4 The step will be marked with a check when it is complete. Clear Assignments to Inlets You can reassign an inlet by clicking it, selecting Clear inlet assignment, and assigning the inlet to the selected step. 38

39 Chapter 4: Customize a Protocol Clear Assignments to Experiment Chambers Clear Assignments to Experiment Chambers To reassign a chamber to a different step, you must first clear the existing assignment to the chamber. 1 Select the step assigned to the chamber you want to clear. 2 Click any chamber and select Clear assignment. This will clear all the chambers assigned to that step and any following steps. In the following example, if you want to assign E5 to the last mix step, select the PCR step. Click chamber E5. Click step for chamber you want to reassign. Click chamber assigned to the step and select Clear assignment. Clear the chamber assignment for the PCR step (chambers E3, E4, and E5 will be cleared). Reassign chambers E3 and E4 to the PCR step. Chamber E5 can then be assigned to the mix step. 39

40 Chapter 4: Customize a Protocol Reuse an Inlet Reuse an Inlet The reuse of inlets for different reagent mixes between scripts should be avoided when possible. However, if there are not enough inlets available for your protocol, you can reuse an inlet by assigning it in more than one script. When you click the inlet to assign it, the menu displays a message to inform you that the inlet is already being used. If you are using the same reagent in more than one step within a single script, you can assign the reagent mix from a list of reagent mixes being used in the script by clicking in the Reagent Mix field, then selecting the Reagent Inlet Reuse tab. IMPORTANT If you are using different reagents when you reuse an inlet, the C1 system will perform additional washing to reduce the residual amount of the previous reagent; however, this will not completely remove the previous reagent from the inlet. Care should be taken to avoid reuse of inlets with incompatible reagents. You must remove all remaining reagent from a reused inlet before loading the next reagent. 40

41 Chapter 5: Manage Reagents In the Protocol Recipe tab and the Reagent Mix Library, you can define the reagents and reagent mixes that are used in your protocol. A reagent is a single component of a reagent mix. A primary reagent mix is assigned to an inlet and can contain secondary reagent mixes, which appear in the Protocol Recipe tab as separate preparatory steps. Protocol Recipe Tab In the Protocol Recipe tab, you can review and modify the reagents used in the scripts. You can also create new definitions for any undefined reagent mixes. Select a script to display the reagent mixes used in each step of the script. Selected script Reagent mixes used in the script 41

42 Chapter 5: Manage Reagents Protocol Recipe Tab Reagent Mixes Pane The Reagent Mixes pane lists the reagent mixes used in each step of the selected script. It also lists any secondary reagent mixes that need to be prepared in advance. For example, the RNA Seq Lysis Mix with Diluted RNA Spikes requires Diluted RNA spikes and the Array Control RNA Spikes to be prepared in tubes. The RNA Seq Lysis mix with Diluted RNA Spikes is therefore considered a primary reagent mix with the Array Control RNA Spikes and RNA Seq Diluted RNA Spikes being the required secondary reagent mixes. Primary reagent mix Secondary reagent mixes Reagent Mix Definition Pane 42

43 Chapter 5: Manage Reagents Protocol Recipe Tab In the Reagent Mix Definition pane, you can: Change the name of the reagent mix. Add or modify special instructions, such as preparation instructions. Add, delete, or import reagent components. Add, delete, or import reagent components (see Add, Import, or Remove Reagent Components on page 45). Adjust concentration and volume values (see Adjust Concentrations and Volume Values on page 46). Modifying the concentration and volume values of a reagent mix does not change the reagent mix stored in the library. Any changes to the mix apply only to this step. Enter the part number, vendor, kit information, and any comments for the reagent component. Modify an Assigned Reagent Mix 1 In the Protocol Recipe tab, click a script to show the reagent mix used in each step. 2 Click a step in the Reagent Mixes pane to modify the reagent(s) used in that step. 3 Modify the Reagent Mix Definition as described in Reagent Mix Definition Pane on page If you want to save the modified reagent mix to the user library using a new name, click Save. 43

44 Chapter 5: Manage Reagents Protocol Recipe Tab Assign a Different Reagent Mix 1 In the Protocol Recipe tab, click a script to show the reagent mix used in each step. 2 Click a step in the Reagent Mixes pane to modify the reagent(s) used in that step. 3 Click Find, then select a reagent mix from the library, then click OK. 4 If desired, modify the Reagent Mix Definition, as described in Reagent Mix Definition Pane on page If you want to save the modified reagent mix to the user library using a new name, click Save. Define a New Reagent Mix If a step does not have a defined reagent mix assigned to it, you can add the definition in the Protocol Recipe tab. An undefined reagent mix has no name or components. 1 In the Protocol Recipe tab, click a step in the Reagent Mixes pane that has an undefined reagent mix. 2 Define the reaction mix as described in Reagent Mix Definition Pane on page If you want to use this reagent mix in other protocols, click Save to add the reagent mix to the User Library. 4 If you added a new reagent mix in step 2: a Define the reaction mix as described in Reagent Mix Definition Pane on page 42. b If desired, save the reagent mix. 44

45 Chapter 5: Manage Reagents Protocol Recipe Tab Add, Import, or Remove Reagent Components 1 Add reagent components, either by: Clicking (New) and selecting New Reagent or New Reagent Mix. This adds a new, blank row to the bottom of the list, or, Clicking (Import) and selecting Import Reagent or Import Reagent Mix from the reagent library. NOTE If you add or import a reagent mix, it will appear below the selected step in the Reagent Mixes pane as a secondary reagent mix. 2 Select a unit for measuring the stock concentration. To remove reagent components, click (Delete Reagent). 45

46 Chapter 5: Manage Reagents Protocol Recipe Tab Adjust Concentrations and Volume Values The Protocol Recipe tab provides a way to calculate mix volumes of the reagents needed to achieve a desired concentration for the assigned inlet(s). The mix volume calculator treats any reagent component without a desired tube or chamber concentration as a buffer for diluting the reagent mix. If the desired concentration is not specified for more than one reagent component, the volume needed to dilute the reagent mix is split evenly across those components. The calculator will also assign a stock concentration value to any reagent component which has a desired concentration (tube or chamber) but no defined stock concentration. To adjust the concentrations and volume values: 1 Select the reagent mix in the Protocol Recipe tab. 2 Enter the stock concentration for the reagent component. 3 Enter either the desired tube or chamber concentration. 4 Tube concentration is the desired concentration in the final mix loaded onto the Open App IFC. Chamber concentration is the desired concentration in the assigned reaction chamber(s). 5 Click to calculate the volumes of the reagents in the mix needed to meet the desired tube or chamber concentration. The calculator allows you to specify the total mix prep volume, and dispensing precision for the inlet. Concentrations that are outside of the desired concentration range by more than 10% are indicated in red. 46

47 Chapter 5: Manage Reagents Reagent Mix Library Reagent Mix Library The Reagent Mix Library (Settings > Reagent Mix Library) contains both the Fluidigm-defined and user-defined reagent mixes that can be used in your scripts. IMPORTANT If you are planning to share your protocol on Script Hub, you must define all reagent mixes used in all the scripts. 47

48 Chapter 5: Manage Reagents Reagent Mix Library Add a New Reagent Mix to the Reagent Library 1 Open the Reagent Mix Library, by selecting Settings > Regent Mix Library, then select the User Library tab. 2 In the Reagent Mixes pane, click (New) to add a new reagent mix. 3 Enter a name for the reagent mix and any special instructions (for example, instructions for preparing the reagents in tubes). 4 In the Reagent Mix pane, add the reagent components for the mix by: Clicking (New) and selecting New Reagent or New Reagent Mix, or, Clicking (Import) and selecting Import Reagent or Import Reagent Mix. 48

49 Chapter 5: Manage Reagents Reagent Mix Library 5 Enter or modify the stock concentration, desired tube or chamber concentration, preparation volume, part number, vendor, kit information, and any comments for the reagent component. 6 If you added a new reagent mix as a secondary component, it will be displayed as a link. Click this link to view and enter or modify the details of the secondary reaction mix. 7 To remove reagent components, click (Delete Reagent). Export the User Library You can export the user-defined reagents for use on another computer running C1 Script Builder : 1 In the Reagent Mix Library window, select the User Library tab. 2 Click Export. The user library is exported as an.xml file. 49

50 Chapter 5: Manage Reagents Reagent List Import a User Library IMPORTANT Importing a library will overwrite any reagent mixes with the same name. To import a user library: 1 In the Reagent Mix Library window, select the User Library tab. 2 Click Import. Navigate to and select the library you wish to import. Reagent List The Reagent List window lists the reagents needed for the protocol, along with part numbers and stock concentrations. The Fluidigm Reagents tab indicates which of the Fluidigm-supplied kits contain one or more of the reagent components. The Other Reagents tab lists the non-fluidigm reagents used in the protocol. Open the Reagent List by selecting Protocol > Reagent List. You can export a tab to a Microsoft Excel spreadsheet by selecting the tab and clicking Export. 50