Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1

Protocol 1: Prep Sample (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Hybridize Probes (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Hybridize Probes for Stranded Total RNA Prep v1.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Calculate Hybridize Probe Master Mix

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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.::DB1 (uL):: = 3.6 * step.::Total Samples:: ; step.::DP1 (uL):: = 1.2 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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
  DB1 (uL)	Numeric		Decimal Places Displayed = 0
  DP1 (uL)	Numeric		Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = Add 4 µl Hybridize Probe Master Mix to each well.
  Thermal Cycler Program	Text		Default = HYB_DP1
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Reaction volume is 15 µl u 95° for 2 minutes Decrease 0.1°C per second until the temperature reaches 37°C Hold at 37°C
  Total Samples	Numeric		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 2: Deplete rRNA (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Deplete rRNA v1.0

• Step Type = No Outputs

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Calculate rRNA Depletion Master Mix

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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.::RDB (uL):: = 4.8 * step.::Total Samples:: ; step.::RDE (uL):: = 1.2 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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 rRNA Depletion Master Mix to each well.
  RDB (uL)	Numeric		Decimal Places Displayed = 0
  RDE (uL)	Numeric		Decimal Places Displayed = 0
  Thermal Cycler Program	Text		Default = RNA_DEP
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = RNA_DEP program on the thermal cycler:Choose the preheat lid option and set to 100°C Reaction volume is 20 µl 37°C for 15 minutes Hold at 4°C
  Total Samples	Numeric		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 3: Remove Probes (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Remove Probes v1.0

• Step Type = No Outputs

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Calculate Removal Master Mix

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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.::PRB (uL):: = 7.7 * step.::Total Samples:: ; step.::PRE (uL):: = 3.3 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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 10 µl Probe Removal Master Mix to each well.
  PRB (uL)	Numeric		Decimal Places Displayed = 0
  PRE (uL)	Numeric		Decimal Places Displayed = 0
  Thermal Cycler Program	Text		Default = PRB_REM
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = PRB_REM program on the thermal cycler: Choose the preheat lid option and set to 100°C Reaction volume is 30 µl 37°C for 15 minutes 70°C for 15 minutes Hold at 4°C
  Total Samples	Numeric		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 4: Clean Up RNA (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Clean Up RNA v1.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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 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: Fragment and Denature RNA (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Fragment and Denature RNA v1.0

• Step Type = No Outputs

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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 8.5 µl EPH3 to each well.
  Thermal Cycler Program	Text		Default = DEN_RNA
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Reaction volume is 17 µl 94°C for 2 minutes Hold at 4°C

• Step File Placeholders

  • Log - Automatically attached

• 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 6: Synthesize First Strand cDNA (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Synthesize First Strand cDNA v1.0

• Step Type = No Outputs

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Calculate First Strand Synthesis Master Mix

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

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}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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	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 7: Synthesize Second Strand cDNA (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus 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 Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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	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 Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Adenylate 3ʹ Ends (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Adenylate 3ʹ Ends - RNA Prep v1.0

• Step Type = No Outputs

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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	Default = Choose the preheat lid option and set to 100°C Reaction volume is 30 µl 37°C for 30 minutes 70°C for 5 minutes 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

Step 2: Ligate Anchors (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)

• Master Step Name = Ligate Anchors - RNA Prep v1.0

• Step Type = No Outputs

• Reagent Kits

  • Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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
  Add Anchors Instruction Notes	Multiline Text		Default = Volume for Sample Input <= 100 ng (µl) 1. Add 2.5 (µl) RSB 2. Add 2.5 (µl) RNA Index Anchors 3. Add 2.5 (µl) LIGX Volume for Sample Input > 100 ng (µl) 1. Add 0 (µl) RSB 2. Add 5 (µl) RNA Index Anchors 3. Add 2.5 (µl) LIGX
  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	Default = Choose the preheated lid option and set to 100°C Reaction volume is 38 µl 30°C for 10 minutes 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

Step 3: Clean Up Fragments (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus 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 Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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 Total RNA Prep Ligation with Ribo-Zero Plus 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 Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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	Presets 15 13 10 Default = 15
  Recommendations for PCR Cycle Number	Multiline Text	Read Only	Default = Use 15 cycles for 10 ng of input DNA Use 13 cycles for 100 ng of input DNA Use 10 cycles for 1000 ng of input DNA
  Thermal Cycler Program	Text		Default = PCR
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Reaction volume is 50 µl u 98°C for 30 seconds X cycles of: 98°C for 10 seconds 60°C for 30 seconds 72°C for 30 seconds 72°C for 5 minutes Hold at 4°C for ≤ 16 hours

• 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 Total RNA Prep Ligation with Ribo-Zero Plus 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 Total RNA Prep, Ligation with Ribo-Zero Plus

    • Supplier = Illumina

    • Website = www.illumina.com

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

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}"

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