TruSeq Small RNA v1.0
Overview
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
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
The version of Ligate Adapters master step name may be different depending on the version of IPP installed.
Automations
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
HML Volume (ul)
Numeric
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
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: 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
Naming Convention = {SubmittedSampleName}
Reagent Kits
SuperScript II Reverse Transcriptase
Supplier = Life Technologies
Catalog Number = 18064-014
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
The version of Perform Reverse Transcription master step name may be different depending on the version of IPP installed.
Automations
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
Dilute 25mM dNTP mix to 12.5mM (per library): 0.5ul of 25mM dNTP Mix and 0.5ul of Ultrapure water.
Toggle Switch
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
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: 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
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
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
The version of Amplify Libraries master step name may be different depending on the version of IPP installed.
Automations
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
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Placement Pattern = Column
Destination Containers
Tube
Add Labels
Label Groups
TruSeq Small RNA
Record Details
Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
Ultrapure water Volume (ul)
Numeric
Decimal Places Displayed = 2
PML Volume (ul)
Numeric
Decimal Places Displayed = 2
RP1 Volume (ul)
Numeric
Decimal Places Displayed = 2
Add 2ul of chosen RPIX to each sample.
Toggle Switch
Default = None Set
Step File Placeholders
Log File - Automatically attached
Log File - 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 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
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
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
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
BioAnalyzer DNA High Sensitivity Chip
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
>=
<=
=
!=
Criteria 1 - Source Data Field
Text Dropdown
Presets
Concentration
Conc. Units
Number of Peaks found
Peak 1 Size - bp
Peak 1 Conc.
Peak 1 Molarity
Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity
Peak 3 Size - bp
Peak 3 Conc.
Peak 3 Molarity
Peak 4 Size - bp
Peak 4 Conc.
Peak 4 Molarity
Peak 5 Size - bp
Peak 5 Conc.
Peak 5 Molarity
Number of Regions found
Region 1 Average Size - bp
Region 1 Conc.
Region 1 Molarity
Region 2 Average Size - bp
Region 2 Conc.
Region 2 Molarity
Region 3 Average Size - bp
Region 3 Conc.
Region 3 Molarity
Region 4 Average Size - bp
Region 4 Conc.
Region 4 Molarity
Region 5 Average Size - bp
Region 5 Conc.
Region 5 Molarity
Criteria 1 - Threshold Value
Numeric
Decimal Places Displayed = 2
Criteria 2 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
!=
Criteria 2 - Source Data Field
Text Dropdown
Presets
Concentration
Conc. Units
Number of Peaks found
Peak 1 Size - bp
Peak 1 Conc.
Peak 1 Molarity
Peak 2 Size - bp
Peak 2 Conc.
Peak 2 Molarity
Peak 3 Size - bp
Peak 3 Conc.
Peak 3 Molarity
Peak 4 Size - bp
Peak 4 Conc.
Peak 4 Molarity
Peak 5 Size - bp
Peak 5 Conc.
Peak 5 Molarity
Number of Regions found
Region 1 Average Size - bp
Region 1 Conc.
Region 1 Molarity
Region 2 Average Size - bp
Region 2 Conc.
Region 2 Molarity
Region 3 Average Size - bp
Region 3 Conc.
Region 3 Molarity
Region 4 Average Size - bp
Region 4 Conc.
Region 4 Molarity
Region 5 Average Size - bp
Region 5 Conc.
Region 5 Molarity
Criteria 2 - Threshold Value
Numeric
Decimal Places Displayed = 2
Use strict matching for Bioanalyzer results
Toggle Switch
Default = None Set
Step File Placeholders
Bioanalyzer Input File - Automatically attached
Bioanalyzer Input 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 = Hide
File Attachment Method = Auto
Table Columns - Global Fields
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Derived Sample
Molarity (nM)
Numeric
Decimal Places Displayed = 2
Derived Sample
Sample Name
Built-in
Measurement
BA Sample Name
Text
Measurement
Concentration
Numeric
Decimal Places Displayed = 2
Measurement
Conc. Units
Text
Measurement
Molarity (nM)
Numeric
Decimal Places Displayed = 2
Measurement
Number of Peaks found
Numeric
Decimal Places Displayed = 0
Measurement
Number of Regions found
Numeric
Decimal Places Displayed = 0
Measurement
Peak 1 Conc.
Numeric
Decimal Places Displayed = 2
Measurement
Peak 1 Molarity
Numeric
Decimal Places Displayed = 2
Measurement
Peak 1 Size - bp
Numeric
Decimal Places Displayed = 0
Measurement
Peak 2 Conc.
Numeric
Decimal Places Displayed = 2
Measurement
Peak 2 Molarity
Numeric
Decimal Places Displayed = 2
Measurement
Peak 2 Size - bp
Numeric
Decimal Places Displayed = 0
Measurement
Peak 3 Conc.
Numeric
Decimal Places Displayed = 2
Measurement
Peak 3 Molarity
Numeric
Decimal Places Displayed = 2
Measurement
Peak 3 Size - bp
Numeric
Decimal Places Displayed = 0
Measurement
Peak 4 Conc.
Numeric
Decimal Places Displayed = 2
Measurement
Peak 4 Molarity
Numeric
Decimal Places Displayed = 2
Measurement
Peak 4 Size - bp
Numeric
Decimal Places Displayed = 0
Measurement
Peak 5 Conc.
Numeric
Decimal Places Displayed = 2
Measurement
Peak 5 Molarity
Numeric
Decimal Places Displayed = 2
Measurement
Peak 5 Size - bp
Numeric
Decimal Places Displayed = 2
Measurement
Region 1 Average Size - bp
Numeric
Decimal Places Displayed = 0
Measurement
Region 1 Conc.
Numeric
Decimal Places Displayed = 2
Measurement
Region 1 Molarity
Numeric
Decimal Places Displayed = 2
Measurement
Region 2 Average