YourSeq (FT & 3’DGE) Strand-SpecificmRNA Library Prep(version A2)

Catalog No. 23001, 23002, 23003, 23004

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TABLE OF CONTENTS

Page

Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 3Choose Full-Transcript (FT) or 3’ Digital Gene Expression (3’DGE) Sequencing . 4
Protocols
Section A: RNA Fragmentation and cDNA Priming . . . . . . . . . . . . . . . . . . . . . . . 6
Section B: cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Section C: 5’-Adapter Sequence Addition  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Section D-CDI: Enrichment and Indexing CDI . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section D-UDI: Enrichment and Indexing UDI  . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section D: Enrichment and Indexing cont. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section E: Final Library Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  12
Section F: Library QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  13
Appendix
Sequencing Guidelines  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Troubleshooting Guide  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  15
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

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Overview
The YourSeq Library Prep workflow takes advantage of the natural phenomenon of
DNA breathing to eliminate several steps which add time and cost to traditional RNA
library prep protocols. DNA breathing is the opening and closing of DNA due to thermal
fluctuations. During library prep, first-strand cDNA synthesis creates a DNA:RNA
hybrid molecule which undergoes DNA breathing at the terminal end. YourSeq takes
advantage of openings at the terminal end of this DNA:RNA hybrid to bind the 5-prime
adapter to the cDNA in a process called “Breath Capture.” This allows the enzymaticsynthesis of a second strand of DNA without removal of RNA.
The workflow also implements strictly-enforced strand specificity as a second adapter
is added by a DNA dependent DNA polymerase (DNA Pol I). As it primes off of the DNA
strand of the DNA-RNA hybrid, it cannot prime off of the RNA strand.
YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kits can be used to prepare
samples for either conventional full-transcript (FT) or 3’-Digital Gene Expression (3’DGE)
sequencing. Conventional full-transcript (FT) RNA-seq yields sequencing reads that
map to the entire expressed transcriptome. The 3’ Digital Gene Expression (3’-DGE)
option creates reads mapping just at the 3’-end of the transcript. The reduced complexity associated with 3’-DGE confers the advantage of requiring a quarter to a third lesssequencing depth, greatly reducing cost.
YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kits can be ordered witheither combinatorial dual indexes or unique dual indexes. Combinatorial dual-indexing
kits allow you to choose from 96 or 384 unique index combinations. Unique dual-indexing kits allow the multiplexing of either 24 or 96 samples at a time without using any
index twice. This can help avoid Illumina index hopping, or the incorrect assignment oflibraries to the expected index.

Product

FormatCatalog No.

YourSeq (FT & 3’DGE) Strand-SpecificmRNA Library Prep 24 CDI

24 rxnCombinatorial Dual Indexes

23001

YourSeq (FT & 3’DGE) Strand-SpecificmRNA Library Prep 96 CDI

96 rxnCombinatorial Dual Indexes

23002

YourSeq (FT & 3’DGE) Strand-SpecificmRNA Library Prep 24 UDI

24 rxnUnique Dual Indexes

23003

YourSeq (FT & 3’DGE) Strand-SpecificmRNA Library Prep 96 UDI

96 rxnUnique Dual Indexes

23004

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1

Kit Components and Storage
YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kits are for research use
only. Not for use in diagnostic procedures. All components are guaranteed stable for6 months from date of receipt when stored properly. Store the Carboxyl Beads at 4°C
after first use. All other components store at -20 °C.
Catalog no.

23001

23002

23003

23004

Storage

Indexing

CDI

CDI

UDI

UDI

Reactions

24

96

24

96

Carboxyl Beads

1.5 ml6 ml

1.5 ml6 ml

4°C

FT Priming Mix

75 μl

300 μl75 μl300 μl-20°C

DGE Priming Mix

75 μl

300 μl75 μl300 μl-20°C

Control mRNA

5 μl

5 μl

5 μl

5 μl

-20°C

First Strand Mix

135 μl540 μl135 μl540 μl-20°C

RT enzyme

15 μl

60 μl

15 μl

60 μl

-20°C

Breath Capture Mix

175 μl690 μl

175 μl690 μl-20°C

5-Prime Adapter

120 μl

480 μl120 μl

480 μl-20°C

DNA Pol I

10 μl

30 μl

10 μl

30 μl

-20°C

PCR Mix

264 μl1056 μl

264 μl1056 μl-20°C

Phusion Pol

8 μl

24 μl

8 μl

24 μl

-20°C

Nuclease-Free Water

1.8 ml

1.8 ml

1.8 ml

1.8 ml-20°C

10 mM Tris HCl

1.8 ml5 ml

1.8 ml

5 ml

-20°C

Carboxyl Beads Buffer

1.8 ml6 ml

1.8 ml

6 ml

-20°C

YourSeq Index i5-A

30 μl

120 μl-20°C

YourSeq Index i5-B

30 μl

120 μl-20°C

YourSeq Index i5-C

30 μl

120 μl-20°C

YourSeq Index i5-D

30 μl

120 μl-20°C

YourSeq i7 1-24 Index Set

1 plateReagent

YourSeq i5 1-24 Index Set
YourSeq i7 1-96 Index Set

1 plateYourSeq i5 1-96 Index Set

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2

1 plate-20°C

1 plate-20°C
1 plate-20°C

1 plate-20°C

Additional materials• Nanodrop™, Qubit™ or equivalent method to determine DNA concentration• Agarose gel electrophoresis equipment (If checking library post amplification)• 2% agarose gel (If checking library post amplification)• Agilent TapeStation or BioAnalyzer (if checking library post clean-up)• 80% ethanol• Vortexer• Thermal cycler• Multichannel pipetter (20 - 200 µl)• Multichannel pipetter (1 - 10 µl)• Filter tips for pipettes• 0.2 ml 8-well PCR strip tubes with caps
• Microcentrifuge for 8-well PCR strip tubes with caps
• 500 ul or 1.5 ml microcentrifuge tubes for master mixes• 96-Well Magnetic Separator for microtiter plates, ex. MagWell Magnetic Separator
96, EdgeBio, cat#57624.

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3Choose Full-Transcript (FT) or 3’ Digital Gene Expression(3’DGE) Sequencing
The YourSeq (FT & 3’DGE) Strand-Specific mRNA Library Prep Kits come with sufficient
reagents for two types of RNA-seq libraries. If you are unsure which is best for yourneeds please read the descriptions below to determine which to use. Note that otherthan the priming mix used in Section A, Step 1, the two protocols are identical.
Full Transcript (FT) Sequencing
The Full Transcript (FT) sequencing option is conventional Full Transcript RNA sequencing and is the common type of RNA sequencing that most people are familiar with.
Conventional full-transcript (FT) coverage RNA-seq yields sequencing reads that map
to the entire expressed transcriptome. This is achieved through the combination of
random RNA fragmentation and subsequent random priming during the cDNA synthesis
step. The result is sequencing reads that, when mapped to the reference transcriptome
or genome, tile across the exons of expressed transcripts. With conventional RNA-seq,
the number of reads mapping to a gene is a function of transcript abundance and transcript length. A long gene, therefore, will have more reads mapping to it than a short
gene of similar transcript abundance.
Use Full-transcript RNA sequencing for:• Differential gene expression for non-annotated genomes• Genotyping and detection of exonic polymorphisms• Splice variant analysis• Detection of gene fusions abundance• Detection of gene fusions abundance.

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4

3’ Digital Gene Expression (3’DGE) Sequencing
The 3’ Digital Gene Expression (3’-DGE) option is optimized for differential gene expression analyses, where the main goal is to identify genes and pathways that are up- or
down- regulated in response to some experimental condition.
Rather than use random priming, the cDNA synthesis step for 3’-DGE RNA-seq libraries
uses an adapter with an oligo(dT) to prime off the transcript’s poly(A) tail. This meansthat 3’-DGE reads tend to map only to the 3’-end of the transcript. Since there is a theoretical 1-to-1 correlation between transcript molecule and cDNA molecule, transcript
length is irrelevant and the number of reads mapping to a gene is solely a function oftranscript abundance. A long gene, therefore, will have the same number of reads mapping to it as will a short gene of similar transcript abundance.
The reduced complexity associated with 3’-DGE confers one of the biggest advantages.
Transcript counting means that less sequencing depth is needed for a robust analysis
compared to conventional RNA-seq. Also, paired-end sequencing does not add value
for 3’-DGE. Therefore, a researcher who might sequence conventional RNA-seq libraries at 20-30 million pairs of paired-end reads per sample can reduce cost by sequencing those same samples as 3’-DGE libraries at 3-10 million single reads per sample.
Use 3’DGE RNA sequencing for:• Differential gene expression for well-annotated genomes• To improve annotation by identifying expressed genes and their 3’-UTRs• Genotyping and detection of polymorphisms in the 3’-UTR• Detection and characterization of alternative polyadenylationwww.activemotif.com

5Assay Protocol
Read the entire protocol before use.
Section A: RNA Fragmentation and cDNA Priming
1.

For each mRNA Sample, dilute 5-100 ng mRNA with Nuclease-Free Water fora total volume of 7.5 µl in a 0.2 ml PCR strip reaction tube. Note that total RNA
contains approximately 3-5% mRNA. For 5-100 ng mRNA we recommend startingwith at least 100 ng - 5 µg of total RNA and doing either poly(A) enrichment or ribosomal depletion of the total RNA. For further guidance contact technical support.
Optional Controls: It’s recommended to include one positive control reaction
consisting of 1 µl of the included Control mRNA diluted with 6.5 µl Nuclease-Free
Water and one negative control reaction consisting of 7.5 µl Nuclease-Free Water.
Reagent

mRNA Sample

Positive Control

Negative ControlmRNA

5-100 ng

1 μl-

Nuclease-free water

up to 7.5 μl

6.5 μl7.5 μlFT Priming Mix
OR*
DGE Priming Mix

2.5 μl

2.5 μl

2.5 μlTotal Volume

10 μl

10 μl

10 μl

2.

Add 2.5 µl Priming Mix (FT or DGE, see Page 4) to the 7.5 µl diluted mRNA & mix
well by pipetting.

3.

Briefly centrifuge reaction tubes to ensure that all of each sample is at the bottomof its tube.

4.

Incubate in thermal cycler for fragmentation and first-strand priming using thefragmentation program:
Step

Temperature

Incubation time

1

25°C

1 sec

2

94°C

1.5 min

3

30°C

1 min

4

20°C

4 min

5

20°C

Hold

Do not put on ice. Proceed directly to Section B.
Important: Place Carboxyl Beads at room temperature for at least 30 min before use.
Carboxyl Beads are light sensitive and should be protected from light when not used.
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6Section B: cDNA Synthesis
1.

Make enough cDNA Synthesis Cocktail for all samples. For each sample, add 0.5µl RT Enzyme + 4.5 µl First Strand Mix into a single microcentrifuge tube (Calculate
using 10% additional volume).

2.

Add 5 µl cDNA Synthesis Cocktail to each Fragmented/Primed mRNA Sample (from
Step A.4) for a total reaction volume of 15 µl. Mix well by pipetting. Briefly centrifuge reaction tubes to ensure that all of each sample is at the bottom of its tube.
Reagent

3.

Volume (per reaction)

cDNA Synthesis Cocktail

5 μl

Fragmented/Primed mRNA (from Step A.4)

10 μl

Total Volume

15 μl

Incubate in thermal cycler for the reverse transcription program as shown below.
Step

Temperature

Incubation time

1

25°C

10 min2

42°C

50 min3

50°C

10 min4

70°C

10 min

5

4°C

Hold

4.

Resuspend Carboxyl Beads (at room temperature) by vortexing.

5.

Add 22.5 µl Carboxyl Beads to each sample. Mix well by pipetting.

6.

Let beads in samples stand 5 minutes at room temperature.

7.

Place tubes on magnetic rack and remove supernatant.

8.

Without resuspending pellet, wash beads twice on magnetic stand.
a. Add 200 µl of 80% ethanol.
b. Pipette up and down 5 times.
c. Remove and discard ethanol.
d. Repeat steps a-c.
After removing ethanol from second wash: spin tubes for 15 seconds, place onmagnetic rack, and remove residual supernatant with a 20 µl pipette. Allow pellets
to dry for 2 min.
Warning: Avoid significantly over-drying beads; otherwise, beads may clump andbe difficult to homogenize.
Note: The protocol can be paused at any point where the beads are in 80% Ethanol at the second wash. Simply store the strip tubes/plates at -20ºC, even for an
extended period of many weeks, if necessary.
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7Section C: 5’-Adapter Sequence Addition
1.

Thaw 5-Prime Adapter and bring to room temperature. Shake and briefly centrifuge tube.

2.

With tubes on magnetic rack, add 4 µl room-temperature 5-Prime Adapter to eachdry bead pellet. It is not necessary to resuspend the beads at this point.

3.

Make enough Breath Capture Cocktail for all samples. For each sample, add 0.25
µl DNA Pol I + 5.75 µl Breath Capture Mix into a single microcentrifuge tube (Calculate using 10% additional volume). Pipette up and down to mix.

4.

Add 6 µl Breath Capture Cocktail to each bead pellet for a total reaction volume of
10 µl. Mix well and resuspend the beads by pipetting.

5.

Incubate in thermal cycler at 25ºC for 15 minutes.
Step

Temperature

Incubation time

1

25°C

15 min6.

Add 15 μl of Carboxyl Beads Buffer to each sample. Mix well and resuspend thebeads by pipetting.

7.

Let resuspended beads stand 5 minutes at room temperature.

8.

Place tubes on magnetic rack and remove supernatant.

9.

Without resuspending pellet, wash beads twice on magnetic stand.
a. Add 200 µl of 80% ethanol.
b. Pipette up and down 5 times.
c. Remove and discard ethanol.
d. Repeat steps a-c.
After removing ethanol from second wash: spin tubes for 15 seconds, place onmagnetic rack, and remove residual supernatant with a 20 µl pipette. Allow pellets
to dry for 2 min.
Warning: Avoid significantly over-drying beads; otherwise, beads may clump andbe difficult to homogenize.
Note: The protocol can be paused at any point where the beads are in 80% Ethanol at the second wash. Simply store the strip tubes/plates at -20ºC, even for an
extended period of many weeks, if necessary.

10. Resuspend pellet in 10 μl 10 mM Tris HCl.
11. Repeat steps 6-9.
12. Resuspend pellet in 12 μl 10 mM Tris HCl.
13. Place tubes on magnetic rack and transfer 10 μl of the cDNA-containing supernatant to fresh tubes. Be careful to avoid bead carryover.

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8For Combinatorial Dual-Indexing (CDI):
Proceed to Section D-CDI, page 9.
For Unique Dual-Indexing (UDI):
Proceed to Section D-UDI, page 10.
Section D-CDI: Enrichment and Indexing, Combinatorial
Dual-Indexing (CDI)YourSeq (FT & 3’DGE) Strand-Specific Library Prep 24 CDI: The 24 rxn kit contains 24
YourSeq Index i7 (1-24) oligos and 4 YourSeq Index i5 (A,B,C, or D) oligos. For eachsample you’ll be pairing one Index i7 (1-24) oligo with one Index i5 (A,B,C, or D)
oligo. There are 96 possible combinations of i7 and i5.
YourSeq (FT & 3’DGE) Strand-Specific Library Prep 96 CDI: The 96 rxn kit contains 96
YourSeq Index i7 (1-24) oligos and 4 YourSeq Index i5 (A,B,C, or D) oligos. For eachsample you’ll be pairing one Index i7 (1-96) oligo with one Index i5 (A,B,C, or D)
oligo. There are 384 possible combinations of i7 and i5.
Note: Set up enrichment at room temperature. Hot start is not necessary.
1.

Make enough Enrichment PCR Cocktail for all samples, adding 0.2 µl Phusion
Polymerase + 7.8 µl PCR Mix into a single tube for each sample (Calculate using
10% additional volume). Pipette up and down to mix.

2.

For each sample, add 8 µl of the Enrichment PCR Cocktail to an empty tube of a
PCR strip.

3.

Add 1 µl of YourSeq i7 Index oligo and 1 µl of a YourSeq i5 (A,B,C,D) Oligo to each
PCR tube so that no combination is used twice.

4.

Add the 10 µl of cDNA Supernatant (from Step C.13) to each PCR tube for a total
reaction volume of 20 µl. Mix well by pipetting.

Reagent

Volume (per reaction)

Enrichment PCR Cocktail

8 μl

YourSeq Index i7 oligo

1 μl

YourSeq Index i5 A,B,C, or D oligo

1 μl

cDNA Supernatant

10 μl

Total Volume

20 μl

Proceed to Section D: Enrichment and Indexing cont., page 11.
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9Section D-UDI: Enrichment and Indexing, Unique Dual-Indexing (UDI)YourSeq (FT & 3’DGE) Strand-Specific Library Prep 24 UDI: The 24 rxn kit contains 24
YourSeq Index i7 (1-24) oligos and 24 YourSeq Index i5 (1-24) oligos. For each reaction you’ll be pairing one Index i7 (1-24) oligo with one Index i5 (1-24) oligo, neverusing any index twice. There are 24 unique dual-index pairs.
YourSeq (FT & 3’DGE) Strand-Specific Library Prep 96 UDI: The 96 rxn kit contains
96 YourSeq Index i7 (1-96) oligos and 96 YourSeq Index i5 (1-96) oligos. For eachsample you’ll be pairing one Index i7 (1-96) oligo with one Index i5 (1-96) oligo.
There are 96 unique dual-index pairs.
Note: Set up enrichment at room temperature. Hot start is not necessary.
1.

Make enough Enrichment PCR Cocktail for all samples, adding 0.2 µl Phusion
Polymerase + 7.8 µl PCR Mix into a single tube for each sample (Calculate using
10% additional volume). Pipette up and down to mix.

2.

For each sample, add 8 µl of the Enrichment PCR Cocktail to an empty tube of a
PCR strip.

3.

Add 1 µl of a YourSeq i7 Index oligo and 1 µl of a YourSeq i5 Oligo to each PCR tube
so that no index oligo is used more than once.

4.

Add the 10 µl of cDNA Supernatant (from Step C.13) to each PCR tube for a total
reaction volume of 20 µl. Mix well by pipetting.

Reagent

Volume (per reaction)

Enrichment PCR Cocktail

8 μl

YourSeq Index i7 oligo

1 μl

YourSeq Index i5 oligo

1 μl

cDNA Supernatant

10 μl

Total Volume

20 μl

Proceed to Section D: Enrichment and Indexing cont., page 11.

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10Section D: Enrichment and Indexing cont.
5.

Optional: Transfer 10 µl of the 20 µl enrichment PCR mix from Step 4 to a second strip of PCR tubes. This can be done without changing tips after mixing. The
second set of PCR reactions can be stored at -20ºC for later use if additional PCR
cycles are required.

6.

Incubate in thermal cycler for enrichment and adapter extension as shown below.
Depending on your particular samples the number of cycles may be more or less.
Start with 14 cycles.
Step

Temperature

Incubation time

Cycles1

98°C

30 sec1

98°C

10 sec65°C

30 sec72°C

30 sec

3

72°C

5 min

4

4°C

Hold

2

14

1

7.

Optional: Run 2 µl of PCR reaction on a 2% Agarose gel to check amplification.
See examples of FT & 3’DGE libraries for positive (Control mRNA) and negative(Nuclease-Free Water) controls below:

8.

If desired amplification is obtained, proceed to Section E: Final Library Cleanup.
Otherwise, use the extra PCR reactions (from Step 5) with an increased or decreased number of cycles.
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11Section E: Final Library Cleanup
1.

Resuspend fresh Carboxyl Beads by vortexing.

2.

Add 1.2 volumes of resuspended Carboxyl Beads to each sample (i.e. 9.6 µl Carboxyl Beads for 8 µl of enrichment product). Mix well by pipetting.

3.

Let beads in samples stand 5 minutes at room temperature.

4.

Place tubes on magnetic rack and remove supernatant.

5.

Without resuspending pellet, wash beads twice on magnetic stand.
a. Add 200 µl of 80% ethanol.
b. Pipette up and down 5 times.
c. Remove and discard ethanol.
d. Repeat steps a-b.
After removing ethanol from second wash: spin tubes for 15 seconds, place onmagnetic rack, and remove residual supernatant with a 20 µl pipette. Allow pellets
to dry for 2 min.
Warning: Avoid significantly over-drying beads; otherwise, beads may clump andbe difficult to homogenize.

6.

Resuspend pellet in 10 µl 10mM Tris HCl.

7.

Add 12 μl of Carboxyl Beads Buffer to each sample. Mix well and resuspend thebeads by pipetting.

8.

Let beads in samples stand 5 min at room temperature.

9.

Place tubes on magnetic rack and remove supernatant.

10. Without resuspending pellet, wash beads twice on magnetic stand.
a. Add 200 µl of 80% ethanol.
b. Pipette up and down 5 times.
c. Remove and discard ethanol.
d. Repeat steps a-b.
After removing ethanol from second wash: spin tubes for 15 seconds, place onmagnetic rack, and remove residual supernatant with a 20 µl pipette. Allow pellets
to dry for 2 min.
Warning: Avoid significantly over-drying beads; otherwise, beads may clump andbe difficult to homogenize.
11. Resuspend pellet in 12 µl 10mM Tris HCl.
12. Place tubes on magnetic rack and transfer 10 μl of the library-containing supernatant to fresh tubes. Be careful to avoid bead carryover.

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12Section F: Library QC
You may evaluate libraries to confirm sufficient recovery from beads and theabsence of adapter contamination. If adapter contaminants are observed, an additional wash of the pooled libraries can be done to ensure removal of any remaining adapter contaminants.
Below are example libraries created using 5 μl of the included Control mRNA andfollowing either the Full Transcript or 3’ Digital Gene Expression protocol. Libraries
were run on an Agilent TapeStation with the D1000 Screen Tape Assay.

Example Full-Transcript (FT) Library

Example Digital Gene Expression (3’DGE) Library

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13

Appendix
Section G: Sequencing Guidelines
Combinatorial Dual Indexed (CDI) Libraries
Libraries from the combinatorial dual-indexing (CDI) version of the kit can be sequenced
as either single-read (Read 1) or paired-end reads (Read 1 + Read 2), with a unique
combination of Index 1 (i7) and Index 2 (i5) per sample.
The 24 reaction, CDI version of the kit comes with 24 Index 1 (i7) adapters and 4 Index 2(i5) adapters. The 96 reaction, CDI version of the kit comes with 96 Index 1 (i7) adaptersand 4 Index 2 (i5) adapters.
Adapter sequences for your kit can be downloaded from the product page at:https://www.activemotif.com/catalog/1358/yourseq-mrna-library-prep#documents
Illumina’s dual-indexing sequencing workflow requires that the reverse complement of
the Index 2 (i5) adapter sequence be entered into the Sample Sheet for some systems.
Use the correct adapter sequence for your instrument as shown in the spreadsheet.

Unique Dual-Indexed (UDI) Libraries
Libraries from the Unique dual-indexing version (UDI) of the kit can be sequenced aseither single-read (Read 1) or paired-end reads (Read 1 + Read 2), with a unique Index 1(i7) and unique Index 2 (i5) per sample.
The 24 reaction, UDI version of the kit comes with 24 Index 1 (i7) adapters and 24
YourSeq Index 2 (i5) adapters, for 24 unique dual-index pairs. The 96 reaction, UDI
version of the kit comes with 96 Index 1 (i7) adapters and 96 Index 2 (i5) adapters, for
96 unique dual-index pairs. No index should be used more than one time. This indexing
strategy is used to combat index hopping. Index hopping is a phenomenon associatedwith Illumina sequencers that utilize patterned flow cells (including HiSeq X/3000/4000
and NovaSeq) whereby a small percentage of reads get assigned to the wrong barcode. Unique dual indexing allows the removal during demultiplexing of unexpectedcombinations caused by index hopping. As a result, you only keep the reads actuallyassociated with your samples.
Adapter sequences for your kit can be downloaded from the product page at:https://www.activemotif.com/catalog/1358/yourseq-mrna-library-prep#documents
Illumina’s dual-indexing sequencing workflow requires that the reverse complement of
the Index 2 (i5) adapter sequence be entered into the Sample Sheet for some systems.
Use the correct adapter sequence for your instrument as shown in the spreadsheet.
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14Troubleshooting Guide
Problem/question

Possible cause

Recommendation

Low library yields

Not enough PCR
cyclesIncrease number of library amplfication PCR cycles.

mRNA input too low

Increase amount of input.

Bioanalyzer peakaround 70 bp

Indexing primers
carried over fromlibrary amplification

Indexing primers can cluster but cannot be sequenced.
Generally not an issue if peak is a small fraction compared to main library peak.

Bioanalyzer peakaround 140 bp

Adapter dimers

Adapter dimers contain complete adapter sequences
and so can cluster on flow cell and sequence. If ratioof adapter dimer peak is low compared to main librarypeak, do nothing. If the peak is high percentage of main
library peak, cleanup the library again.

Second Bioanalzyer
peak approximately 2X
the main library peak

Overcycling

Reduce number of library amplification PCR cycles.

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Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of
the numbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150 Toll Free: 877 222 9543
Carlsbad, CA 92008, USA
Direct: 760 431 1263
E-mail: tech_service@activemotif.com
Fax: 760 431 1351
Active Motif Europe
Waterloo Atrium
Drève Richelle 167 – boîte 4
BE-1410 Waterloo, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Active Motif China
889 Wanhangdu Road, Suite 1602B Direct: (86)-21-20926090
Jingan District
Cell Phone: 18521362870
Shanghai, China 200040
E-mail: techchina@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter and notification of our upcoming products• Share your ideas and results with us• View our job opportunities
Don’t forget to bookmark our site for easy reference!

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