# Illumina Stranded mRNA Prep Ligation 1.1

## Overview

The Illumina Stranded mRNA Prep Ligation workflow includes the following functionality.

* Preconfigured protocols that convert the messenger (mRNA) in total RNA into up to 384 dual-indexed libraries. Depletion reagents bind and deplete abundant transcripts from total RNA. Reverse transcription then converts the remaining RNA into cDNA, while subsequent ligation and amplification steps add adapters for clustering and sequencing on an Illumina system.
* Automated calculation of sample and buffer volumes.
* Automated calculation or display of reagents at every step in the protocol.
* Automatic step transition when required.
* Automatic placement of samples when necessary.
* Automated assignment of QC Pass/Fail, based on user-selected threshold values.
* A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

## Protocol 1: Prep Sample (Illumina Stranded mRNA Prep Ligation v1.1)

Protocol Type = Sample Prep

**Next Steps Configuration**

<figure><img src="/files/AimWISZ30x1fNhkFavLO" alt=""><figcaption></figcaption></figure>

### Step 1: Deplete mRNA (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Deplete mRNA v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/dyZS2fsIPqfD2859HITQ" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate Fragmentation Master Mix</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon entry

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::Nuclease-free ultrapure water (uL):: = 10.5 * step.::Total Samples:: ; step.::EPH3 (uL):: = 10.5 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Sample and Water Volume</summary>

* Trigger Location = Record Details
* Trigger Style = Manual button

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'output.::SampleVolume (uL):: = (step.::Final Diluted Concentration (ng/uL):: * step.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; output.::Nuclease-free Ultrapure Water (uL):: = step.::Final Volume (uL):: - output.::SampleVolume (uL)::' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                                        | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
  | ----------------------------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | EPH3 (uL)                                             | Numeric        |                |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |
  | Final Diluted Concentration (ng/uL)                   | Numeric        | Required Field | <ul><li>Default = 10</li><li>Range = 10 To 1000</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |
  | Final Volume (uL)                                     | Numeric        | Required Field | <ul><li>Default = 25</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
  | Instruction Notes                                     | Multiline Text | Read Only      | <ul><li>Default =<br>Final Diluted Concentration should be between 10-1000 ng/uL.<br><br>Enter in Starting Concentration before clicking on Calculate Sample and Water Volumes.<br><br>Add 19 µl Fragmentation Master Mix to each well.</li></ul>                                                                                                                                                                                                                                                                                                                           |
  | Nuclease-free ultrapure water (uL)                    | Numeric        |                | <ul><li>Decimal Places Displayed = 0</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
  | Thermal Cycler Program for Capture                    | Text           |                | <ul><li>Default = mRNA\_CAP</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
  | Thermal Cycler Program for Elute                      | Text           |                | <ul><li>Default = mRNA\_ELT</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
  | Thermal Cycler Program for Fragment and Denature mRNA | Text           |                | <ul><li>Default = DEN94\_8</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |
  | Thermal Cycler Program Notes                          | Multiline Text | Read Only      | <ul><li>Default =<br>mRNA\_CAP program on the thermal cycler:<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 50 µl u 65°C for 5 minutes<br>4°C for 30 seconds<br>23° for 5 minutes<br>Hold at 23°C<br><br>mRNA\_ELT program on the thermal cycler:<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 25 µl<br>80°C for 2 minutes<br>Hold at 25°C<br><br>DEN94\_8 program on the thermal cycler:<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 19 µl<br>94°C for 8 minutes<br>Hold at 4°C</li></ul> |
  | Total Samples                                         | Numeric        |                | <ul><li>Default = 0</li><li>Decimal Places Displayed = 0</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**                     | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ---------------------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name                     | Built-in       |             |                                           |
    | Container      | LIMS ID (Container)                | Built-in       |             |                                           |
    | Container      | Well                               | Built-in       |             |                                           |
    | Derived Sample | Nuclease-free Ultrapure Water (uL) | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | Sample Name                        | Built-in       |             |                                           |
    | Derived Sample | SampleVolume (uL)                  | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | Starting Concentration (ng/uL)     | Numeric        |             | Decimal Places Displayed = 0              |
    | Project        | Project Name                       | Built-in       |             |                                           |

### Step 2: Synthesize First Strand cDNA (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Synthesize First Strand cDNA v1.0
* Step Type = No Outputs
* Reagent Kits
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/KQLSpe1aEv9KgRWH4gdh" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate First Strand Synthesis Master Mix</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon entry

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::FSA (uL):: = 9 * step.::Total Samples:: ; step.::RVT (uL):: = 1 * step.::Total Samples:: ' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**               | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                                                                                          |
  | ---------------------------- | -------------- | ----------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | FSA (uL)                     | Numeric        |             | Decimal Places Displayed = 0                                                                                                                                                       |
  | Instruction Notes            | Multiline Text | Read Only   | Default = Add 8 uL of First Strand Synthesis Master Mix to each sample.                                                                                                            |
  | RVT (uL)                     | Numeric        |             | Decimal Places Displayed = 0                                                                                                                                                       |
  | Thermal Cycler Program       | Text           |             | Default = FSS                                                                                                                                                                      |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 25 µl<br>25°C for 10 minutes<br>42°C for 15 minutes<br>70°C for 15 minutes<br>Hold at 4°C</p> |
  | Total Samples                | Numeric        |             | Default = 0                                                                                                                                                                        |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 3: Synthesize Second Strand cDNA (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Synthesize Second Strand cDNA v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP, 5 ml
    * Supplier = Beckman Coulter
    * Catalog Number = A63880
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/pbBrlQiTx8fFOl2zAjhp" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**               | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                                      |
  | ---------------------------- | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------ |
  | 80% EtOH Prep Date           | Date           |             |                                                                                                                                |
  | Thermal Cycler Program       | Text           |             | Default = SSS                                                                                                                  |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 40°C<br>Reaction volume is 50 µl u 16°C for 1 hour<br>Hold at 4°C</p> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

## Protocol 2: Library Prep (Illumina Stranded mRNA Prep Ligation v1.1)

Protocol Type = Library Prep

**Next Steps Configuration**

<figure><img src="/files/BhwCPIGIVUqwLQwmUhZc" alt=""><figcaption></figcaption></figure>

### Step 1: Adenylate 3ʹ Ends (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Adenylate 3ʹ Ends - RNA Prep v1.0
* Step Type = No Outputs
* Reagent Kits
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/JWl5r5Ap8mA02kJGRbON" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**               | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                                                                  |
  | ---------------------------- | -------------- | ----------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | Instruction Notes            | Multiline Text | Read Only   | Default = Add 12.5 µl ATL4 to each well.                                                                                                                   |
  | Thermal Cycler Program       | Text           |             | Default = ATAIL                                                                                                                                            |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 30 µl<br>37°C for 30 minutes<br>70°C for 5 minutes<br>Hold at 4°C</p> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 2: Ligate Anchors (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Ligate Anchors - RNA Prep v1.0
* Step Type = No Outputs
* Reagent Kits
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/do28oFsEdqA6y6aTJEbf" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**               | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                                              |
  | ---------------------------- | -------------- | ----------- | -------------------------------------------------------------------------------------------------------------------------------------- |
  | Instruction Notes            | Multiline Text | Read Only   | Default = Add 5 µl STL to each well.                                                                                                   |
  | Thermal Cycler Program       | Text           |             | Default = LIG                                                                                                                          |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheated lid option and set to 100°C<br>Reaction volume is 38 µl<br>30°C for 10 minutes<br>Hold at 4°C</p> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 3: Clean Up Fragments (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Clean Up Fragments - RNA Prep v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP, 5 ml
    * Supplier = Beckman Coulter
    * Catalog Number = A63880
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/V1EhQfXMvGVisa0ScsDI" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**     | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | 80% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 4: Amplify Library (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Amplify Library - RNA Prep v1.0
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}-{AppliedReagentLabels}
* Reagent Kits
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/0zt3zVSA6TvSYtA5aJ86" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Add Labels

* Label Groups
  * IDT for Illumina RNA UD Indexes Set A Ligation
  * IDT for Illumina RNA UD Indexes Set A-D Ligation
  * IDT for Illumina RNA UD Indexes Set B Ligation
  * IDT for Illumina RNA UD Indexes Set C Ligation
  * IDT for Illumina RNA UD Indexes Set D Ligation
  * Illumina RNA UD Indexes Set A B C D Ligation
  * Illumina RNA UD Indexes Set A Ligation
  * Illumina RNA UD Indexes Set B Ligation
  * Illumina RNA UD Indexes Set C Ligation
  * Illumina RNA UD Indexes Set D Ligation

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                       | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                                                                                                                                                                                                                                |
  | ------------------------------------ | -------------- | -------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
  | PCR Cycles                           | Text Dropdown  | Custom Entries | <ul><li><p>Presets</p><ul><li>15</li><li>13</li><li>10</li></ul></li><li>Default = 15</li></ul>                                                                                                                                                                          |
  | Recommendations for PCR Cycle Number | Multiline Text | Read Only      | <ul><li>Default =<br>Use 15 cycles for 10 ng of input DNA<br>Use 13 cycles for 100 ng of input DNA<br>Use 10 cycles for 1000 ng of input DNA</li></ul>                                                                                                                   |
  | Thermal Cycler Program               | Text           |                | <ul><li>Default = PCR</li></ul>                                                                                                                                                                                                                                          |
  | Thermal Cycler Program Notes         | Multiline Text | Read Only      | <ul><li>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 50 µl u 98°C for 30 seconds<br>X cycles of:<br>98°C for 10 seconds<br>60°C for 30 seconds<br>72°C for 30 seconds<br>72°C for 5 minutes<br>Hold at 4°C for ≤ 16 hours</li></ul> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 5: Clean Up Library (Illumina Stranded mRNA Prep Ligation v1.1)

* Master Step Name = Clean Library v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP, 5 ml
    * Supplier = Beckman Coulter
    * Catalog Number = A63880
  * Illumina Stranded mRNA Prep, Ligation
    * Supplier = Illumina
    * Website = [www.illumina.com](https://help.connected.illumina.com/clarity-lims/library-prep/other-rna-protocols/www.illumina.com)

#### Automations

<figure><img src="/files/ufzs4vd6wE7S2CX68ZxH" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**     | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | 80% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |


---

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