TruSeq RNA Exome v1.0

Overview

TruSeq RNA Exome v1.0 includes the following functionality:

• Preconfigured TruSeq RNA Exome v1.0 protocol that explains how to convert total RNA into a library of template molecules of known strand origin, then capture the coding regions of the transcriptome using the reagents provided in Illumina® TruSeq® RNA Access Library Prep kits. The resulting library is suitable for subsequent cluster generation and 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: TruSeq RNA Exome v1.0

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Fragment RNA (TruSeq RNA Exome v1.0)

• Master Step Name = Fragment RNA v2.0
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {SubmittedSampleName}
• Reagent Kits

  • TruSeq RNA Library Prep for Enrichment

    • Supplier = Illumina
    • Catalog Number = 20020189
    • Website = www.illumina.com

Automations

Dilute Total RNA

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

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 = Row
• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Desired Diluted Concentration (ng/uL)	Numeric	Required Field	Decimal Places Displayed = 1
  Thermal Cycler Program	Text		Default = Elution 2 - Frag - Prime

• Step File Placeholders

  • Log File - Automatically attached
• Sample Table

  • Sample Display Default = Expand
  • 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	Buffer Volume (ul)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Volume (ul)	Numeric		Decimal Places Displayed = 2
    Project	Project Name	Built-in

Step 2: Synthesize First Strand cDNA (TruSeq RNA Exome v1.0)

• Master Step Name = First Strand cDNA Synthesis v2.0
• Step Type = No Outputs
• Reagent Kits

  • SuperScript II Reverse Transcriptase

    • Supplier = Life Technologies
    • Catalog Number = 18064-014
    • Website = http://www.lifetechnologies.com/order/catalog/product/18064014
  • TruSeq RNA Library Prep for Enrichment

    • Supplier = Illumina
    • Catalog Number = 20020189
    • 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	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Thermal Cycler Program	Text Dropdown	Custom Entries	Presets Synthesize 1st Strand 1stSS Default = Synthesize 1st Strand

• Step File Placeholders

  • Log File - Automatically attached
• Sample Table

  • Sample Display Default = Expand
  • 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 3: Synthesize Second Strand cDNA (TruSeq RNA Exome v1.0)

• Master Step Name = Second Strand cDNA Synthesis v2.0
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {SubmittedSampleName}
• Reagent Kits

  • AMPure XP Beads

    • Supplier = Beckman Coulter Genomics
    • Catalog Number = A63881
    • Website = https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm
  • TruSeq RNA Library Prep for Enrichment

    • Supplier = Illumina
    • Catalog Number = 20020189
    • 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 = Row
• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  80% EtOH Prep Date	Date

• Step File Placeholders

  • Log File - 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	Sample Name	Built-in
    Project	Project Name	Built-in

Step 4: Adenylate 3' Ends (TruSeq RNA Exome v1.0)

• Master Step Name = Adenylate 3' Ends v2.0
• Step Type = No Outputs
• Reagent Kits

  • TruSeq RNA Library Prep for Enrichment

    • Supplier = Illumina
    • Catalog Number = 20020189
    • 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 = Row
• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  ATL to add to each well (uL)	Numeric		Default = 12.5
  Comment	Multiline Text
  RSB to add to each well (uL)	Numeric		Default = 2.5

• Step File Placeholders

  • Log File - 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	Sample Name	Built-in
    Project	Project Name	Built-in

Step 5: Ligate Adapters (TruSeq RNA Exome v1.0)

• Master Step Name = Ligate Adapters v2.0
• Step Type = Add Labels
• Derived Sample Generation = Fixed, 1
• Naming Convention = {SubmittedSampleName}
• Reagent Kits

  • AMPure XP Beads

    • Supplier = Beckman Coulter Genomics
    • Catalog Number = A63881
    • Website = https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm
  • TruSeq RNA Library Prep for Enrichment

    • Supplier = Illumina
    • Catalog Number = 20020189
    • Website = www.illumina.com

Automations

Set Next Step - Advance

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

Copy to Output

• Trigger Location = Not Used

Set Next Step & Copy to Input

• Trigger Location = Not Used

Queue/Ice Bucket

• 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	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Add Labels

• Label Groups

  • TruSeq RNA Single Indexes (12 indexes, 24 samples) Set A
  • TruSeq RNA Single Indexes (12 indexes, 24 samples) Set B

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Round 1 EtOH to each well (uL)	Numeric		Default = 42
  Round 1 RSB to each well (uL)	Numeric		Default = 52.5
  Round 2 EtOH to each well (uL)	Numeric		Default = 50
  Round 2 RSB to each well (uL)	Numeric		Default = 22.5
  1 RSB to add to each well (uL)	Numeric		Default = 2.5
  2 LIG to add to each well (uL)	Numeric		Default = 2.5
  3 RNA Adapters to add to each well (uL)	Numeric		Default = 2.5
  80% EtOH Prep Date	Date

• Step File Placeholders

  • Log File - Automatically attached
• Sample Table

  • Sample Display Default = Expand
  • 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	Reagent Name	Built-in
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 6: Enrich DNA Fragments (TruSeq RNA Exome v1.0)

• Master Step Name = Enrich DNA Fragments (TruSeq RNA Exome v1.0.6)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {SubmittedSampleName}
• Reagent Kits

  • AMPure XP Beads

    • Supplier = Beckman Coulter Genomics
    • Catalog Number = A63881
    • Website = https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm
  • TruSeq RNA Library Prep Enrichment Component, Box 1

    • Supplier = Illumina
    • Catalog Number = 20020490
    • Website = www.illumina.com
  • TruSeq RNA Library Prep for Enrichment

    • Supplier = Illumina
    • Catalog Number = 20020189
    • 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 = Row
• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Thermal Cycler Program	Text		Default = PCR
  80% EtOH Prep Date	Date

• Step File Placeholders

  • Log File - Automatically attached
• Sample Table

  • Sample Display Default = Expand
  • 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 7: Bioanalyzer QC (Library Validation) (TruSeq RNA Exome v1.0)

• Master Step Name = Bioanalyzer QC (Library Validation) v2.0
• Step Type = Standard QC
• Measurement Generation = Fixed, 1
• Naming Convention = {InputItemName} Bioanalyzer

Automations

Generate Bioanalyzer Input file

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

Parse Bioanalyzer XML and assign QC flags

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

Set Next Step - Output PASS/FAIL

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

Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations

• Trigger Location = Not Used

Parse Bioanalyzer XML, Calculate nM and assign QC flags

• Trigger Location = Not Used

Parse Bioanalyzer XML, Copy nM and Assign QC flags

• Trigger Location = Not Used

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers
  • Well Sort Order = Row
• Sample Table (Column Headers)

  Category	Field Name