# Illumina RNA Prep with Enrichment (L) Tagmentation v1.1 ## Overview The Illumina RNA Prep with Enrichment (L) Tagmentation workflow includes the following functionality. * Preset Illumina RNA Prep with Enrichment (L) Tagmentation protocols that support the generation of enriched libraries for dual-indexed, paired-end sequencing. * 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. ## Protocol 1: Prep Sample (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) Protocol Type = Sample Prep **Next Steps Configuration**

### Step 1: Denature RNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Denature RNA v1.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Calculate Sample and Water Volume

* 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 %}

Set Next Step - Advance

* 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 %}

#### Queue * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * 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 | | | #### 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** | | ----------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------- | | Final Diluted Concentration (ng/uL) | Numeric | Required Field |

Default = 10
Range = 10 To 100

Default = 8.5

Default = Enter Final Diluted Concentration and Starting Concentration before clicking on the Calculate Sample and Water Volume button.

Default = DEN\_RNA

Default =
Choose the preheat lid option and set to 100°C
Reaction volume is 17 µl
65°C for 5 minutes
Hold at 4°C

| * 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 RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Synthesize First Strand cDNA v1.0 * Step Type = No Outputs * Reagent Kits * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Calculate First Strand Synthesis Master Mix

* 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 %}

Set Next Step - Advance

Default =
Choose the preheat lid option and set to 100°C
Reaction volume is 25 µl
25°C for 10 minutes
42°C for 15 minutes
70°C for 15 minutes
Hold at 4°C

| | 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 RNA Prep with Enrichment (L) Tagmentation 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 RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Set Next Step - Advance

Default =
Choose the preheat lid option and set to 40°C
Reaction volume is 50 µl u 16°C for 1 hour
Hold at 4°C

| * 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 RNA Prep with Enrichment (L) Tagmentation v1.1) Protocol Type = Library Prep **Next Steps Configuration**

### Step 1: Tagment cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Tagment cDNA v1.0 * Step Type = No Outputs * Reagent Kits * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Calculate Tagmentation Master Mix - RNA Prep

* 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.::TB1 (uL):: = 11.5 * step.::Total Samples:: ; step.::EBLTL (uL):: = 11.5 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 14.5 * step.::Total Samples::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}

Set Next Step - Advance

Default =
Choose the preheat lid option and set to 100°C
Reaction volume is 50 µl u 55°C for 5 minutes
Hold at 10°C

| | 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 2: Amplify Tagmented cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Amplify Tagmented cDNA v1.0 * Step Type = Add Labels * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName}-{AppliedReagentLabels} #### Automations

Calculate PCR Master Mix - RNA Prep

* 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.::EPM (uL):: = 23 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 23 * step.::Total Samples::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}

Set Next Step - Advance

#### 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 | | | #### Placement = Enabled * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Placement Pattern = Column * Destination Containers * 96 well plate #### Add Labels * Label Groups * IDT for Illumina DNA-RNA UD Indexes Set A Tagmentation * IDT for Illumina DNA-RNA UD Indexes Set B Tagmentation * IDT for Illumina DNA-RNA UD Indexes Set C Tagmentation * IDT for Illumina DNA-RNA UD Indexes Set D Tagmentation * Illumina DNA-RNA UD Indexes Set A Tagmentation * Illumina DNA-RNA UD Indexes Set B Tagmentation * Illumina DNA-RNA UD Indexes Set C Tagmentation * Illumina DNA-RNA UD Indexes Set D Tagmentation #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------------------- | -------------- | -------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | EPM (uL) | Numeric | | Decimal Places Displayed = 0 | | Instruction Notes | Multiline Text | Read Only | Default = Add 40 µl PCR Master Mix to each well. | | Nuclease-free ultrapure water (uL) | Numeric | | Decimal Places Displayed = 0 | | PCR Cycles | Text Dropdown | Custom Entries |

Presets

14 cycles for High-quality RNA with DV200 > 80%
17 cycles for FFPE and RNA with DV200 < 80%

Default =
Choose the preheat lid option and set to 100°C
Reaction volume is 50 µl u 72°C for 3 minutes u 98°C for 3 minutes
X cycles of: u 98°C for 20 seconds u 60°C for 30 seconds
72°C for 1 minute u 72°C for 3 minutes
Hold at 10°C

| | 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: Clean Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Clean Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * 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 RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Set Next Step - Advance

### Step 1: Qubit Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Qubit Library QC v1.0 * Step Type = Standard QC * Measurement Generation = Fixed, 1 * Naming Convention = {InputItemName} #### Automations

Assign QC flags

* 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 -u {username} -p {password} \ script:assignQC \ -i {processURI:v2} \ -log {compoundOutputFileLuid1} \ -qcResult {compoundOutputFileLuid2}" ``` {% endcode %}

Set Next Step - Advance

* Trigger Location = Not Used {% 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 %}

#### 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** | | ------------------------------ | -------------- | ----------- | ----------------------------------------- | | Criteria 1 - Operator | Text | | Default = >= | | Criteria 1 - Source Data Field | Text | | Default = Concentration | | Criteria 1 - Threshold Value | Numeric | | Default = 200 | | Criteria 2 - Operator | Text | | Default = <= | | Criteria 2 - Source Data Field | Text | | Default = Concentration | | Criteria 2 - Threshold Value | Numeric | | Default = 1500 | * Step File Placeholders * Log - Automatically attached * QC Result File - Manually uploaded * Sample Table * Enable QC Flags = Yes * Sample Display Default = Expand * Well Sort Order = Column * File Column Options * File Column Display = Show * File Attachment Method = Manual * 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 | Concentration | Numeric | Required Field | Decimal Places Displayed = 2 | | Derived Sample | Conc. Units | Text | Required Field | | | Derived Sample | Sample Name | Built-in | | | | Measurement | Concentration | Numeric | | Decimal Places Displayed = 2 | | Measurement | Conc. Units | Text | | | | Project | Project Name | Built-in | | | ### Step 2: BioAnalyzer Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = BioAnalyzer Library QC v1.0 * Step Type = Standard QC * Measurement Generation = Fixed, 1 * Naming Convention = {InputItemName} #### Automations

Generate Bioanalyzer Input file

* 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/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ',False,' -h 1 -c LIMSID -pre 'Sample '" ``` {% endcode %}

Parse Bioanalyzer XML and assign QC flags

* 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 -u {username} -p {password} -i {processURI:v2} \ script:parseBioAnalyzer \ -inputFile {compoundOutputFileLuid2} \ -log {compoundOutputFileLuid5} \ -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' \ script:assignQC \ -log {compoundOutputFileLuid6} \ -qcResult {compoundOutputFileLuid7}" ``` {% endcode %}

Set Next Step - Advance

* BioAnalyzer DNA 1000 Chip

#### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------- | | Criteria 1 - Operator | Text Dropdown | Custom Entries |

Presets

Presets

Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity

Presets

Presets

Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity

| | Criteria 2 - Threshold | Numeric | | | | Use strict matching for Bioanalyzer results | Toggle Switch | | Default = None Set | * Step File Placeholders * Bioanalyzer Driver File - Automatically attached * Bioanalyzer Driver File Generation Log File - Automatically attached * Bioanalyzer XML Result File (required) - Manually uploaded * Result File (optional) - Manually uploaded * PDF Summary File (optional) - Manually uploaded * Bioanalyzer XML Parsing Log File - Automatically attached * QC Assignment Log File - Automatically attached * QC Assignment Report - Automatically attached * Sample Table * Enable QC Flags = Yes * Sample Display Default = Expand * Well Sort Order = Column * File Column Options * File Column Display = Show * File Attachment Method = Manual * 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: Dilute Libraries (Illumina RNA Prep Tagmentation (L) with Enrichment v1.0) * Master Step Name = Dilute Libraries v1.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Calculate Sample, RSB, & Final Volumes

* 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):: * output.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; if (step.::Enrichment Plexity:: == 1) {output.::Final Volume (uL):: = 7.5} else {output.::Final Volume (uL):: = 2.5} ; step.::RSB (uL):: = output.::Final Volume (uL):: - output.::SampleVolume (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}

Set Next Step - Advance

Required Field
Custom Entries

Presets

Read Only

Default =
In one well of a new PCR plate, combine the applicable number of 200 ng libraries:
If Enrichment Plexity is 1 then add 7.5 ul of the sample.
If Enrichment Plexity is 3 then add 2.5 ul of each sample to one well with a final volume of 7.5 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 | Final Volume (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | RSB (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 4: Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Illumina Exome Panel * Supplier = Illumina * Catalog Number = 20020183 * Website = [www.illumina.com](https://www.illumina.com) * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Set Next Step - Advance

Default =
Choose the preheat lid option and set to 100°C
Reaction volume is 25 µl u 95°C for 5 minutes
18 cycles of 1 minute each: u 94°C for the first cycle
Decrease 2°C per subsequent cycle
58°C for 90 minutes
Hold at 58°C for ≤ 24 hours

| * Step File Placeholders * Log - Manually uploaded * Sample Table * Sample Display Default = Collapse * Well Sort Order = Row * 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: Capture Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Capture Hybridize Probes v1.0 * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Calculate Elution Master Mix

* 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.::EEL (uL):: = 28.5 * step.::Total Samples:: ; step.::HP3 (uL):: = 1.5 * step.::Total Samples::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}

Set Next Step - Advance

Decimal Places Displayed = 0

Decimal Places Displayed = 0

Default = Add 23 µl Elution Master Mix to each well.

Default =
Choose the preheat lid option and set to 70°C
Reaction volume is 100 µl
Hold at 58°C

Default = 0
Decimal Places Displayed = 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 6: Amplify Enriched Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Amplify Enriched Library v1.0 * Step Type = No Outputs * Reagent Kits * Illumina RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Set Next Step - Advance

Default =
Choose the preheat lid option and set to 100°C
Reaction volume is 50 µl
98°C for 30 seconds
14 cycles of:
98°C for 10 seconds
60°C for 30 seconds
72°C for 30 seconds
72°C for 5 minutes
Hold at 10°C

| * Step File Placeholders * Log - Manually uploaded * 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 7: Clean Up Enriched Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1) * Master Step Name = Clean Up Enriched 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 RNA Prep with Enrichment (L) Tagmentation * Supplier = Illumina * Catalog Number = 16 - 20040536, 96 - 20040537 * Website = [www.illumina.com](https://www.illumina.com) #### Automations

Set Next Step - Advance