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

Set Next Step - Advance

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

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

Set Next Step - Advance

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

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

Set Next Step - Advance

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

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

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

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

Set Next Step - Advance

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

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

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