Illumina Preset Protocol - Illumina Stranded mRNA Prep Ligation

The Illumina Stranded mRNA Prep Ligation workflow includes the following functionality.

• Preconfigured protocols that convert the messenger (mRNA) in total RNA into up to 384 dual-indexed libraries. Depletion reagents bind and deplete abundant transcripts from total RNA. Reverse transcription then converts the remaining RNA into cDNA, while subsequent ligation and amplification steps add adapters for clustering and sequencing on an Illumina system.
• 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.

Protocol 1: Prep Sample (Illumina Stranded mRNA Prep Ligation)

Master Step 1: Deplete mRNA

Step Type: Standard

Input = Total RNA

Output = Hexamer-primed RNA fragments

Reagent Kits

Kit Name

Supplier

Illumina Stranded mRNA Prep, Ligation

Illumina
Queue/Ice Bucket

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column

Placement Pattern

Same Shape
Placement

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Final Diluted Concentration (ng/μl)

Numeric
• Range = 10 - 1000
• Requires user input

 

 

Final Volume (μl)

Numeric
• Requires user input

Default

• 25
   

Total Samples

Numeric
• Calculated = Total Samples + 1

Default

• 0
   

Nuclease-free ultrapure water (µl)

Numeric
• Calculated = 10.5 * Total Samples
   

EPH3 (µl)

Numeric
• Calculated = 10.5 * Total Samples
   

Thermal Cycler Program for Capture

Text

Default

• mRNA_CAP
   

Thermal Cycler Program for Elute

Text

Default

• mRNA_ELT
   

Thermal Cycler Program for Fragment and Denature mRNA

Text

Default

• DEN94_8

 

Multiline Text Fields

Thermal Cycler Program Notes

Multiline Text

Read Only

Default

mRNA_CAP program on the thermal cycler:

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 50 µl u 65°C for 5 minutes.
• 4°C for 30 seconds.
• 23° for 5 minutes.
• Hold at 23°C.

mRNA_ELT program on the thermal cycler:

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 25 µl.
• 80°C for 2 minutes.
• Hold at 25°C.

DEN94_8 program on the thermal cycler:

• Choose the preheat lid option and set to 100°C.
• Reaction volume is 19 µl.
• 94°C for 8 minutes.
• Hold at 4°C.
   

Instruction Notes

Multiline Text

Read Only

Default

• Final Diluted Concentration should be between 10-1000 ng/µl.
• Enter in Starting Concentration before clicking on Calculate Sample and Water Volumes.
• Add 19 µl Fragmentation Master Mix to each well.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Starting Concentration (ng/µl)

Numeric

Requires user input
   

SampleVolume (µl)

  
• Global field - derived sample
• Calculated = (Final Diluted Concentration (ng/µl) * Final Volume (µl))/Starting Concentration (ng/µl)

 

  

Nuclease-free Ultrapure Water (µl)

  
• Global field - derived sample
• Calculated = Final Volume (µl) - Sample Volume (µl)
Automation

Automation Name

Setting

Value

Specification

Calculate Sample and Water Volume

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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 'output.::SampleVolume (uL):: = (step.::Final Diluted Concentration (ng/uL):: * step.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; output.::Nuclease-free Ultrapure Water (uL):: = step.::Final Volume (uL):: - output.::SampleVolume (uL)::' \

-log {compoundOutputFileLuid0}"

Calculate Fragmentation Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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.::Nuclease-free ultrapure water (uL):: = 10.5 * step.::Total Samples:: ; step.::EPH3 (uL):: = 10.5 * step.::Total Samples::' \

-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Synthesize First Strand cDNA

Step Type: No Output

Input = Denatured purified total RNA

Output = First strand complementary DNA (cDNA)

Reagent Kits

Kit Name

Supplier

Illumina Stranded mRNA Prep, Ligation

Illumina
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric

Calculated = Total Samples + 1

 

 

FSA (µl)

Numeric

Calculated = 9 * Total Samples
   

RVT (µl)

Numeric

Calculated = 1 * Total Samples
   

Thermal Cycler Program

Text

Default

• FSS
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 8 µl of First Strand Synthesis Master Mix to each sample.
   

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 u 42°C for 15 minutes.
• 70°C for 15 minutes.
• Hold at 4°C.

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate First Strand Synthesis Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Synthesize Second Strand cDNA

Step Type: Standard

Input = First strand complementary DNA (cDNA)

Output =Blunt-ended, double-stranded cDNA fragments

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Stranded mRNA Prep, Ligation

Illumina

  

Agencourt AMPure XP, 5 ml

Beckman Coulter

A63880
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Row
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep Date

Date

  
   

Thermal Cycler Program

Text

Default

• SSS
 

Multiline Text Fields

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

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 2: Library Prep (Illumina Stranded mRNA Prep Ligation)

Master Step 1: Adenylate 3ʹ Ends - RNA Prep

Step Type: No Output

Input = Blunt fragmented cDNA

Output = cDNA with adenine 3' ends

Reagent Kits

Kit Name

Supplier

Illumina Stranded mRNA Prep, Ligation

Illumina
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• ATAIL
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 12.5 µl ATL4 to each well.
   

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

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Ligate Anchors - RNA Prep

Step Type: No Output

Input = Double stranded cDNA

Output = Double stranded cDNA with ligated anchors

Reagent Kits

Kit Name

Supplier

Illumina Stranded mRNA Prep, Ligation

Illumina
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• LIG
 

Multiline Text Fields

Instruction Notes

Multiline Text

Default

• Add 5 µl STL to each well.
   

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

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Clean Up Fragments - RNA Prep

Step Type: Standard

Input = Double stranded cDNA with ligated anchors

Output = Adapter-ligated fragments

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Stranded mRNA Prep, Ligation

Illumina

  

Agencourt AMPure XP, 5 ml

Beckman Coulter

A63880
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column

Placement Screen

Auto
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep Date

Date

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 4: Amplify Library - RNA Prep

Step Type: Add Labels

Input = Anchor-ligated DNA fragments

Output = Dual-indexed library: DNA fragments with adapters at each end.

Reagent Kits

Kit Name

Supplier

Illumina Stranded mRNA Prep, Ligation

Illumina
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Add Labels

Setting

Value

Label Groups

IDT for Illumina RNA UD Indexes Set A 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
 

IDT for Illumina RNA UD Indexes Set A-D Ligation
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• PCR
   

PCR Cycles

Numeric Dropdown
• 15 [default]
• 13
• 10
   

Instruction Notes

Multiline Text

Default

• Add 5 µl STL to each well.
 

Multiline Text Fields

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.
   

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.

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 5: Clean Library

Step Type: Standard

Input = Dual-indexed library

Output = Purified dual-indexed library

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Stranded mRNA Prep, Ligation

Illumina

  

Agencourt AMPure XP, 5 ml

Beckman Coulter

A63880
Queue/Ice Bucket

Setting

Value

Well Sort Order

Column
Placement

Setting

Value

Destination Containers

96 well plate

Well Sort Order

Column
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep Date

Date

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

 

 

Trigger Style

Automatic upon exit

 

 

 

 

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

End of Workflow