TruSeq Small RNA v1.0 includes the following functionality:
Preconfigured TruSeq Small RNA v1.0 protocol that explains how to prepare total RNA or purified small RNA using a Illumina® TruSeq® Small RNA Library Prep Kit.
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.
It is not required to have the total RNA samples quantified prior to starting this protocol, however it is highly recommended to ensure the starting number of samples.
Protocol 1: TruSeq Small RNA v1.0
Protocol Type = Library Prep
Next Steps Configuration
Step 1: Ligate Adapters (TruSeq Small RNA v1.0)
Master Step Name = Ligate Adapters (TruSeq Small RNA v1.0.10)
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Ligate Adapters master step name may be different depending on the version of IPP installed.
Automations
Calculate 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 = Row
Record Details
Step Data (Master Step Fields)
Step 2: Perform Reverse Transcription (TruSeq Small RNA v1.0)
Master Step Name = Perform Reverse Transcription (TruSeq Small RNA v1.0.10)
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Perform Reverse Transcription master step name may be different depending on the version of IPP installed.
Automations
Calculate 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 = Row
Record Details
Step Data (Master Step Fields)
Step 3: Amplify Libraries (TruSeq Small RNA v1.0)
Master Step Name = Amplify Libraries (TruSeq Small RNA v1.0.10)
Step Type = Add Labels
Derived Sample Generation = Fixed, 1
The version of Amplify Libraries master step name may be different depending on the version of IPP installed.
Automations
Calculate Master Mix
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Add Labels
Label Groups
TruSeq Small RNA
Record Details
Step Data (Master Step Fields)
Step 4: Bioanalyzer QC (Library Validation) (TruSeq Small RNA v1.0)
Master Step Name = Bioanalyzer QC (Library Validation) v2.0
Step Type = Standard QC
Measurement Generation = Fixed, 1
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
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Step 5: Run Gel Electrophoresis and Recover Purified Construct (TruSeq Small RNA v1.0)
Master Step Name = Run Gel Electrophoresis and Recover Purified Construct (TruSeq Small RNA v1.0.10)
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Run Gel Electrophoresis and Recover Purified Construct master step name may be different depending on the version of IPP installed.
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Record Details
Step Data (Master Step Fields)
Step 6: Concentrate Final Library (TruSeq Small RNA v1.0)
Master Step Name = Concentrate Final Library (TruSeq Small RNA v1.0.10)
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Concentrate Final Library master step name may be different depending on the version of IPP installed.
Automations
Calculate Master Mix for Pellet Paint
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
Record Details
Step Data (Master Step Fields)
Step 7: Bioanalyzer QC (Library Validation) (TruSeq Small RNA v1.0)
Master Step Name = Bioanalyzer QC (Library Validation) v2.0
Step Type = Standard QC
Measurement Generation = Fixed, 1
Automations
Generate Bioanalyzer Input file
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Parse Bioanalyzer XML, Calculate nM 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 and assign QC flags
Trigger Location = Not Used
Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations
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
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Group of Defaults
Nextera DNA Flex Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
Nextera Mate Pair Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
Nextera XT DNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
NRCC Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq ChIP-Seq Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Exome Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq Methyl Capture EPIC Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq Rapid Exome Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq RNA Access Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq RNA Exome Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq Small RNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Stranded mRNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Stranded Total RNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Targeted RNA Expression Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSight Myeloid Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSight RNA Fusion Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TSCA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
Step Data
Group of Defaults = TruSeq Small RNA Library Validation
Master Step Fields
Step 8: Normalize Libraries (TruSeq Small RNA v1.0)
Master Step Name = Normalize Libraries 1 v2.0.10
Step Type = Standard
Derived Sample Generation = Fixed, 1
The version of Normalize Libraries 1 master step name may be different depending on the version of IPP installed.
Automations
Normalization Calculations - Option 1
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Remove
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Routing script - Normalize Libraries
Trigger Location = Step
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Record Details
Step Data (Master Step Fields)
Naming Convention = {SubmittedSampleName}
Reagent Kits
T4 RNA Ligase 2, Deletion Mutant
Supplier = Epicentre
Catalog Number = LR2D1132K; LR2D11310K
TruSeq Small RNA Library Prep Kit, Core Solutions
Supplier = Illumina
Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
TruSeq Small RNA Library Prep Kit, Indices A, B, C or D
Supplier = Illumina
Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
Control Types
Human Brain Total RNA
Supplier = Thermo Fisher Scientific
Catalog Number = AM7962
Sample Table
Column Headers
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Expanded View Fields
Decimal Places Displayed = 2
RA5 Volume (ul)
Numeric
Decimal Places Displayed = 2
RNase Inhibitor Volume (ul)
Numeric
Decimal Places Displayed = 2
T4 RNA Ligase 2, Depletion Mutant Volume (ul)
Numeric
Decimal Places Displayed = 2
T4 RNA Ligase Volume (ul)
Numeric
Decimal Places Displayed = 2
10mM ATP Volume (ul)
Numeric
Decimal Places Displayed = 2
Step File Placeholders
Log File - Automatically attached
Log File - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Row
Table Columns - Global Fields
Naming Convention = {SubmittedSampleName}
Reagent Kits
SuperScript II Reverse Transcriptase
Supplier = Life Technologies
Catalog Number = 18064-014
Website =
TruSeq Small RNA Library Prep Kit, Core Solutions
Supplier = Illumina
Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
TruSeq Small RNA Library Prep Kit, Indices A, B, C or D
Supplier = Illumina
Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
Sample Table
Column Headers
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Expanded View Fields
Default = None Set
RNase Inhibitor Volume (ul)
Numeric
Decimal Places Displayed = 2
SuperScript II Reverse Transcriptase Volume (ul)
Numeric
Decimal Places Displayed = 2
5X First Strand Buffer Volume (ul)
Numeric
Decimal Places Displayed = 2
12.5 mM dNTP Mix Volume (ul)
Numeric
Decimal Places Displayed = 2
100mM DTT Volume (ul)
Numeric
Decimal Places Displayed = 2
Step File Placeholders
Log File - Automatically attached
Log File - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Row
Table Columns - Global Fields
Naming Convention = {SubmittedSampleName}
Reagent Kits
TruSeq Small RNA Library Prep Kit, Core Solutions
Supplier = Illumina
Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
Website =
TruSeq Small RNA Library Prep Kit, Indices A, B, C or D
Supplier = Illumina
Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
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 = Hide
File Attachment Method = Auto
Table Columns - Global Fields
Naming Convention = {InputItemName}
ℹ The field value and actual version of the workflows and steps in the routing automation script may be different depending on the version of IPP installed.
Sample Table
Column Headers
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Expanded View Fields
Decimal Places Displayed = 2
Target Normalization (nM)
Numeric
Required Field
Default = 2
Decimal Places Displayed = 2
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Row
Table Columns - Global Fields
ℹ The preset options for Derived Sample Sequencing Instrument may vary depending on the version of the IPP.
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
HML Volume (ul)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
Dilute 25mM dNTP mix to 12.5mM (per library): 0.5ul of 25mM dNTP Mix and 0.5ul of Ultrapure water.
