Protocol for TotalScript™ RNA-Seq Kit

TotalScript RNA-Seq Kit
Cat. No. TSRNA1296 – 6 Reactions
(Contains 1 box of Cat. No. TSCD1296 and 1 box of Cat. No. TSLP1296)
Cat. No. TSRNA12924 – 24 Reactions
(Contains 1 box of Cat. No. TSCD12924 and 1 box of Cat. No. TSLP12924)
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TotalScript RNA-Seq Kit
1. Kit Contents
TotalScript cDNA Kit (Cat. Nos. TSCD1296 / TSCD12924)
Component Name
Volume
6 Reactions 24 Reactions
TotalScript 1st Strand Buffer 15 µl 60 μl
TotalScript Optimized Buffer 15 µl 60 μl
Random Hexamer Primer 6 µl 24 μl
Oligo d(T) Primer 6 µl 24 µl
DTT 21 µl 84 μl
dNTPs 5 µl 12 μl
RiboGuard RNase Inhibitor 6 µl 24 µl
EpiScript Reverse Transcriptase 6 µl 24 µl
TotalScript 2nd Strand Master Mix 150 µl 600 µl
Nuclease-Free Water 1 ml 2 X 1.5 ml
Storage: Store this kit box and its contents at –20°C.
Optional Reagent for TotalScript cDNA Synthesis:
Actinomycin D 250 ng/µl (≈0.2 mM) in DMSO; Actinomycin D can improve the
strandedness of the TotalScript library by ≈2% (Biovision, cat. no. 1036-50).
Cap Color
Clear
TotalScript Library Prep Kit (Cat. Nos. TSLP1296 / TSLP12924)
Component Name
Volume
6 Rxn 24 Rxn
TotalScript Tagment Buffer 60 µl 240 µl
TotalScript Enzyme 6 µl 24 μl
Gap-Fill Buffer 24 µl 96 μl
Gap-Fill Enzyme 6 µl 24 μl
TotalScript PCR Primer Cocktail 6 µl 24 μl
TotalScript Read 1 Sequencing Primer 30 µl 100 µl
TotalScript Read 2 Sequencing Primer 30 µl 100 µl
TotalScript Index Read Sequencing
Primer
30 µl 100 µl
Index 1 6 µl 24 µl
TotalScript Control RNA 10 µl 10 µl
TotalScript Stop Solution 300 µl 1.2 ml
Storage: Store this kit box and its contents at –20°C.
Cap Color
Green
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TotalScript RNA-Seq Kit
Required Reagents for TotalScript Library Prep Kit:
Agencourt AMPure XP Kit (Beckman Coulter, cat. no. A63881)
2X Phusion® High Fidelity PCR Master Mix with HF Buffer (NEB, Cat. No. M0531) or
2X Phusion® High Fidelity PCR Master Mix with GC Buffer (NEB, Cat. No. M0532)
10 mM Tris-HCl, pH 8
Optional Reagent:
TotalScript Index Kit, Cat No. TSIDX12910, 11 indexes
(Refer to Appendix C for Index Sequences)
2. Before starting
DNA-Free RNA
Treat the RNA sample with DNase I to remove all traces of DNA. Then, remove the DNase I
prior to the TotalScript cDNA synthesis procedure.
RNA Quality
For best results, use intact, non-fragmented RNA samples with RIN >7 as assayed using a
Bioanalyzer.
Amount of RNA
TotalScript is optimized for 1-5 ng of total RNA input, without any prior ribosomal
RNA removal or poly(A) enrichment. It is crucial to carefully quantify total RNA prior to
beginning. For optimal results, do not exceed the maximum amount of RNA (5 ng).
cDNA Primer and Buffer Combination
The choice of 1st strand cDNA primer and 1st strand cDNA Buffer to use in the 1st strand
cDNA synthesis procedure (step 3) and the attributes of the resulting TotalScript library
are dependent on the RNA sample.
a) When using Total RNA sample:
Follow the procedure in Part 3.A. For best results use the TotalScript Optimized Buffer.
Then, use these guidelines for the choice of cDNA primer that best meets your needs.
• Oligo (dT) Primer yields 3′-bias libraries with

TotalScript RNA-Seq Kit
3. TotalScript cDNA Synthesis Procedure
Steps 3.A – 3.C use the components of the TotalScript cDNA Kit.
Component Name
Oligo(dT) Primer
Random Primers
RNase-Free Water
1st Strand Buffer or Optimized Buffer
DTT
dNTPs
RiboGuard RNase Inhibitor
EpiScript Reverse Transcriptase
Cap Color
Clear
3.A. Total RNA Samples
For best results use the TotalScript Optimized Buffer for total RNA samples. Use the
following guidelines for the choice of cDNA primer that best meets your needs.
• Oligo (dT) Primer yields 3′-bias libraries with

TotalScript RNA-Seq Kit
Important! If using the Mixed Primer option, dilute the Random Hexamer Primer 1:3 with
nuclease free water.
Note: It is recommended to use the Control RNA as a positive control for library prep. Refer to
Appendix B for Control RNA protocol.
1. Heat denature the RNA and anneal the desired cDNA primers. Combine:
x µl Total RNA (1 ng - 5 ng)
2.5 µl TotalScript Optimized Buffer
x µl cDNA primer:
1 µl of Oligo (dT) Primer OR
1 µl of Random Hexamer Primer OR
2 µl of Mixed Primer (1 µl of Oligo (dT) Primer and 1 µl of 1:3 diluted
Random Hexamer Primer
x µl Nuclease-free water
18 µl total volume
2. Place the samples in a thermocycler with heated lid. Heat for 2 minutes at 65°C,
then hold at 4°C.
3.B. 1st Strand cDNA synthesis
Addition of Actinomycin D to the 1st strand cDNA synthesis reaction is optional. Adding
Actinomycin D can improve the strandedness of the TotalScript library by ≈2%.
1. To each 18 µl reaction from Part 3.A add on ice:
2.5 µl DTT
0.5 µl dNTPs
1 µl RiboGuard RNase Inhibitor
1 µl Actinomycin D (250 ng/µl) or Water
1 µl EpiScript Reverse Transcriptase
24 µl total reaction volume
2. In a thermocycler with heated lid, incubate the samples for:
5 minutes at 25°C
25 minutes at 42°C
15 minutes at 70°C
Hold at 4°C
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TotalScript RNA-Seq Kit
3.C. Second Strand Synthesis
Briefly centrifuge each tube from Part 3.B prior to use.
1. To each 24 µl reaction, add the following and mix on ice:
1 µl DTT
25 µl 2nd Strand Master Mix
50 µl total reaction volume
2. Incubate the reactions for 1 hour at 16°C.
3. Heat inactivate the reactions for 15 minutes at 80°C.
4. Hold at 4°C.
Proceed to TotalScript Library Preparation Procedure (Step 4) or freeze the samples
at –20°C.
4. TotalScript Library Prep Kit Procedure
Steps 4.A – 4.F use components of the TotalScript Library Prep Kit
Component Name
TotalScript Tagment Buffer
TotalScript Enzyme
Gap-Fill Buffer
Gap-Fill Enzyme
TotalScript PCR Primer Cocktail
TotalScript Read 1 Sequencing Primer
TotalScript Read 2 Sequencing Primer
TotalScript Index Read Sequencing Primer
Index 1
TotalScript Control RNA
TotalScript Stop Solution
Cap Color
Green
Required Reagents:
Agencourt XP Kit (Beckman Coulter, cat. no. A63881)
2X Phusion® High Fidelity PCR Master Mix with HF Buffer (NEB, cat. no. M0531) or
2X Phusion® High Fidelity PCR Master Mix with GC Buffer (NEB, cat. no. M0532)
Note: For best results, we recommend non-stick microcentrifuge tubes.
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TotalScript RNA-Seq Kit
4.A. Tagmentation procedure
1. Briefly centrifuge each tube from 3.C. Then, pipette 39 µl of each reaction into a
separate microcentrifuge tube.
To each reaction (39 µl), add on ice:
10 µl TotalScript Tagment Buffer
1 µl TotalScript Enzyme
50 µl total reaction volume
2. In a thermocycler with heated lid incubate the samples for:
5 minutes at 55°C.
Hold at 4°C.
Add 5 μl of TotalScript Stop Solution to each sample. Incubate the samples at room
temperature for 5 minutes. The total reaction volume is now 55 µl.
4.B. Purify the reactions using AMPure XP magnetic beads.
1. Prepare at least 500 µl of fresh 80% ethanol solution for each sample.
2. Add 65 μL AMPure beads to each reaction. Mix well and let stand at room
temperature for 5 minutes.
3. Place the samples on magnetic stand and capture beads for 5 minutes, then remove
unbound material.
4. While still on magnet, wash beads twice with 250 μl of 80% Ethanol.
5. Remove any remaining ethanol and allow the beads to dry for 5 minutes.
6. Remove the tubes from the magnetic stand and resuspend the beads in 15 μl of
Elution Buffer (10 mM Tris-HCl; pH 8).
7. Place the tubes on the magnetic stand and capture the beads with magnet for
5 minutes. The tagmented cDNA is now in the Elution Buffer phase.
8. Transfer 14 μl of the eluted, Tagmented DNA to fresh microcentrifuge tubes at room
temperature.
4.C. Oligo Replacement
Kit components required in Part 4.C
Component Name
Gap-Fill Buffer
Index 1
Gap-Fill Enzyme
Cap Color
Green
1. Briefly centrifuge each tube prior to use. Then, combine at room temperature:
14 µl purified Tagmented DNA from Step 4.B
4 µl Gap-Fill Buffer
1 µl Index 1*
19 µl total volume
* or use an index from the TotalScript Index Kit, Cat. No. TSIDX12910
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TotalScript RNA-Seq Kit
2. Incubate each sample for 1 minute at 45°C
3. Incubate each sample for 30 minutes at 37°C
4. Add 1 μl of Gap-Fill Enzyme.
5. Incubate each sample for 30 minutes at 37°C. Then hold at 4°C.
4.D. Purify the reactions using AMPure XP magnetic beads.
1. Prepare at least 500 µl of fresh 80% ethanol solution for each sample
2. Add 25 μl AMPure beads to each reaction. Mix well and let stand at room
temperature for 5 minutes.
3. Place the tubes on magnetic stand and capture beads for 5 minutes, then remove
the unbound material.
4. While still on the magnetic stand, wash the beads twice with 250 μl 80% ethanol.
5. Remove any remaining ethanol and allow the beads to dry for 5 minutes.
6. Remove the tubes from the magnetic stand and resuspend the beads in 25 μl of
Elution Buffer.
7. Place the tubes on the magnetic stand and capture the beads for 5 minutes. The
tagmented cDNA is now in the Elution Buffer phase.
8. Transfer 24 μl of the eluted, gap-filled DNA to fresh PCR tubes and cool on ice.
4.E. PCR amplification
Additionally required (provided by the user)
2X Phusion® High Fidelity PCR Master Mix with HF Buffer (NEB, cat. no. M0531) or
2X Phusion® High Fidelity PCR Master Mix with GC Buffer (NEB, cat. no. M0532)
1. Briefly centrifuge each tube, then combine on ice:
12 µl Gap-Filled cDNA from Step 4.D*
0.5 µl TotalScript PCR Primer Cocktail
12.5 µl 2X Phusion High-Fidelity PCR Master Mix (HF Buffer or GC Buffer)
25 µl total volume
*Store the remaining Gap-Filled cDNA at –20°C.
2. PCR cycle conditions: The number of PCR cycles is dependent on the input RNA and
the cDNA primer used in 1st-strand cDNA synthesis (Part 3.A):
Input Random Primer Mix dT
1 ng 12 14 16
5 ng 10 12 14
Heat at 95°C for 2 minutes.
Then perform the appropriate number of cycles of:
94°C for 10 seconds
60°C for 30 seconds
72°C for 1 minute
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TotalScript RNA-Seq Kit
4.F. Purify the reactions using AMPure XP magnetic beads.
1. Prepare 500 µl of fresh 80% ethanol solution for each sample
2. Add 23 μl AMPure beads (0.9X) to each reaction. Mix well and let stand at room
temperature for 5 minutes.
Note: To increase library fragment/insert size, 0.7X AMPure beads ratio can be used
instead of 0.9X.
3. Place the tubes on magnetic stand and capture beads for 5 minutes, then remove
the unbound material.
4. While still on magnet, wash beads twice with 250 μl of 80% ethanol.
5. Remove any remaining ethanol and allow the beads to dry for 5 minutes.
6. Remove the tubes from the magnetic stand and resuspend the beads in 25 μl of
Elution Buffer.
7. Place the tubes on the magnetic stand and capture the beads with magnet for
5 minutes. The tagmented cDNA is now in the Elution Buffer phase.
8. Transfer 25 μl of the PCR amplified cDNA to fresh PCR tubes and cool on ice.
5. Library analysis and quantification.
The TotalScript RNA-Seq library can be analyzed using the 2100 Bioanalyzer (Agilent). Use
1 μl of each sample with a 2100 Bioanalyzer High Sensitivity DNA Chip.
The yield of the TotalScript RNA-Seq library can be determined by standard laboratory
methods.
6. Sequencing TotalScript RNA-Seq libraries
If sequencing TotalScript libraries with HiSeq, HiScanSQ, or GAIIx, the TruSeq Dual Index
Sequencing Primer Box must be used:
• TruSeq Dual Index Sequencing Primer Kit, Single Read, Cat. No. FC-121-1003, OR
• TruSeq Dual Index Sequencing Primer Kit, Paired End, Cat. No. PE-121-1003
TruSeq Dual Index Sequencing primers will be used in place of HP6, HP7, and HP8
primer mixes. Alternatively, if TruSeq Dual Index Sequencing Primer Box is not accessible,
sequencing primers provided in the TotalScript kit can be used.
TotalScript Read 1, Read 2, and Index Sequencing Primers are provided as 200X (100 um).
Dilute TotalScript Read 1 Sequencing Primer into HP6.
Dilute TotalScript Read 2 Sequencing Primer into HP7.
Dilute TotalScript Index Read Sequencing Primer into HP8.
TotalScript libraries are compatible with the MiSeq system, and do not require the
included sequencing primers or the TruSeq Dual Index Sequencing Primer Boxes for
sequencing on a MiSeq.
Note: The sequence generated by the Read 1 sequencing primer corresponds to the reverse
complement (antisense) sequence of the original RNA molecule.
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TotalScript RNA-Seq Kit
7. Appendix
Appendix A. Bioanalyzer Profile Of A TotalScript RNA-Seq Library
A TotalScript RNA-Seq library was made from 5 ng of random primed Universal Human
Reference RNA (UHR) using the TotalScript Optimized Buffer as described in Part 3.A of
the procedure.
Appendix B. It is recommended to use the Control RNA as a positive control for library
prep to determine that all the steps of the protocol are followed as directed.
1. Thaw the Control RNA and keep on ice. Remove 1 ul of the Control RNA and place
in a fresh RNase-free tube, then add 99 ul of Nuclease-free water and mix well. Keep
diluted Control RNA on ice.
2. Set up the following reaction, as per step 3.A.1. of the TotalScript cDNA Synthesis
procedure:
2 μl 1:100 diluted Control RNA
2.5 μl TotalScript First-Strand Buffer
1 μl Random Hexamer Primers
12.5 μl Nuclease-free water
18.0 μl Total
3. Place the samples in a thermocycler with a heated lid. Heat for 2 minutes at 65°C,
then hold at 4°C.
4. Proceed to Part 3.B, 1st Strand cDNA synthesis.
5. At Step 4.E. PCR Amplification:
a. Amplify the sample for 12 cycles and purify the reaction as directed in Part 4.F. of
the protocol.
b. Run 1 ul on a bioanalyzer using a high sensitivity chip.
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TotalScript RNA-Seq Kit
Appendix C. TotalScript Index Primer Sequences
Index 1 5′-TAAGGCGA-3′
Index 2 CGTACTAG
Index 3 AGGCAGAA
Index 4 TCCTGAGC
Index 5 GGACTCCT
Index 6 TAGGCATG
Index 7 CTCTCTAC
Index 8 CAGAGAGG
Index 9 GCTACGCT
Index 10 CGAGGCTG
Index 11 AAGAGGCA
Index 12 GTAGAGGA
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