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.
Default =
Final Diluted Concentration should be between 10-1000 ng/uL.
Enter in Starting Concentration before clicking on Calculate Sample and Water Volumes.
Add 19 µl Fragmentation Master Mix to each well.
Nuclease-free ultrapure water (uL)
Numeric
Decimal Places Displayed = 0
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
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
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
Naming Convention = {InputItemName}
Reagent Kits
Agencourt AMPure XP, 5 ml
Supplier = Beckman Coulter
Catalog Number = A63880
Illumina Stranded mRNA Prep, Ligation
Supplier = Illumina
Website =
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
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
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
Thermal Cycler Program
Text
Default = PCR
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
Step 2: Ligate Anchors (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)
Master Step Name = Ligate Anchors - RNA Prep v1.0
Step Type = No Outputs
Reagent Kits
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
Record Details
Step Data (Master Step Fields)
Step 3: Clean Up Fragments (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)
Master Step Name = Clean Up Fragments - RNA Prep v1.0
Step Type = Standard
Derived Sample Generation = Fixed, 1
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
Record Details
Step Data (Master Step Fields)
Step 4: Amplify Library (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)
Master Step Name = Amplify Library - RNA Prep v1.0
Step Type = Add Labels
Derived Sample Generation = Fixed, 1
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
Add Labels
Label Groups
IDT for Illumina RNA UD Indexes Set A Ligation
IDT for Illumina RNA UD Indexes Set A-D Ligation
Record Details
Step Data (Master Step Fields)
Step 5: Clean Up Library (Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus v1.1)
Master Step Name = Clean Library v1.0
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
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
Record Details
Step Data (Master Step Fields)
Naming Convention = {InputItemName}
Reagent Kits
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
Supplier = Illumina
Website =
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
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
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
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
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
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
Reagent Kits
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
Supplier = Illumina
Website =
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
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
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
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
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 =
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
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
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus
Supplier = Illumina
Website =
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
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
Illumina RNA UD Indexes Set A B C D Ligation
Illumina RNA UD Indexes Set A Ligation
Illumina RNA UD Indexes Set B Ligation
Illumina RNA UD Indexes Set C Ligation
Illumina RNA UD Indexes Set D Ligation
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
Thermal Cycler Program
Text
Default = PCR
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
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
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 =
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
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Field Name
Field Type
Options
Additional Options and Dropdown Items
DB1 (uL)
Numeric
Decimal Places Displayed = 0
DP1 (uL)
Numeric
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
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
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
Field Name
Field Type
Options
Additional Options and Dropdown Items
FSA (uL)
Numeric
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Prep Date
Date
Thermal Cycler Program
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default = Add 12.5 µl ATL4 to each well.
Thermal Cycler Program
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Add Anchors Instruction Notes
Multiline Text
Default =
Volume for Sample Input <= 100 ng (µl)
1. Add 2.5 (µl) RSB
2. Add 2.5 (µl) RNA Index Anchors
3. Add 2.5 (µl) LIGX
Volume for Sample Input > 100 ng (µl)
1. Add 0 (µl) RSB
2. Add 5 (µl) RNA Index Anchors
3. Add 2.5 (µl) LIGX
