D-Plex mRNA-seq Kit
mRNA library preparation kit
for Illumina® sequencing
Cat. No. C05030033 (24 rxns)

USER GUIDEV1 05_2021

Please read this manual carefully
before starting your experimentIndexes are not included in this kit and are available
separately. Please check "Required materials notprovided" section.

Summary
Kit Method Overview

4

Introduction

5

Kit Materials

7

Required Materials Not Provided

8

Remarks Before Starting

10

Short Protocol (for experienced users)

16

Detailed Protocol

18

PART A: D-Plex mRNA Capture

19

PART B: D-Plex Library preparation

23

D-Plex mRNA Construct

32

Sequencing Recommendations

33

Data Analysis Recommendations

35

Example of Results

41

Related Products

42

Kit Method Overview
Time Pause

RNA

STEP
1

AAAAA
TTTTT

STEP
2

RNA

RNA
UMI
+
5’ adaptor

rXrXrXrX

1h

AAAAA

RNA fragmentationand dephosphorylation

AAAAAAA...

RNA tailing

1h

Reverse transcription
with template switching

>2h

RNA

STEP
3

STEP
4

mRNA capture

30 minAAAAAAA...V TTTTTTTT...XXXXAAAAAAA
TTTTTTTT

XXXX
XXXX

AAAAAAA
TTTTTTTTPCR forward
PCR reverse

STEP
5

AAAAAAA
TTTTTTTT

XXXX
XXXX

PCR amplification

DNA

4

STEP
6

Library clean-up

STEP
7

Library quality control
and quantification

D-PLEX mRNA-SEQ KIT MANUAL

45 minIntroduction
The Diagenode D-Plex mRNA-seq Library Preparation kit is a tool designed
for the study of the whole coding part of the transcriptome. The present
kit incorporates the unique D-Plex technology to generate directional
mRNA libraries for Illumina sequencing directly from total RNAs.
The D-Plex technology utilizes the innovative capture and amplification
by tailing and switching, a ligation-free method for library preparationand offers key advantages such as:• Ultra-low input capability of the library preparation• Ease of use in a one day, one tube protocol• High quality libraries with higher library complexity than mostof other available library preparation kits for RNA-sequencing
The D-Plex mRNA-seq kit (C05030033) is composed of the D-Plex
mRNA capture module (C05030032) and the D-Plex Total RNA-seq kit(C05030031). The mRNA capture protocol is based on oligo(dT) magnetic
beads to capture poly(A)-tailed RNAs such as messenger RNAs (mRNAs)and some long non-coding RNAs (lncRNAs). The other transcripts present
in the total RNA sample – without poly(A) tails – will not be captured by
the oligo(dT) beads and hence will not be extracted from the total RNA
sample. It should be noted that certain mRNAs from different organisms
do not bear a poly(A) tail and will thus not be captured (e.g. prokaryotes,
FFPE-degraded mRNAs). The D-Plex Total RNA-seq library preparation
protocol delivers a high-sensitivity detection method that incorporates
the widest possible diversity of mRNAs.
The core of the D-Plex technology relies on ligation-free reactions
to attach the Illumina adaptors to both ends of the library construct.
The entire library construction protocol, starting with mRNAs, can becompleted in less than 5 hours with minimal handling steps and handson time. The workflow includes template fragmentation and end repair,polynucleotide tailing, reverse transcription with template-switching,and PCR amplification. The directionality of the template-switching
reaction preserves the strand orientation of the mRNA template, which
thus provides indication on the DNA strand from which the mRNA was5transcribed. Retaining strandness information allows for identificationof antisense transcripts, determination of the transcribed strand of
non-coding RNAs, and measurement of expression levels of coding or
non-coding overlapping transcripts.
For optimal workflow flexibility, D-Plex Unique Dual Indexes Modules(C05030021 and C05030022) are available separately from the library
preparation kit, providing PCR primers for library multiplexing up to 48.
The use of unique dual indexes (UDI) is highly recommended to mitigate
errors introduced by read misassignment, including index hopping
frequently observed with patterned flow cells such as Illumina’s NovaSeq
6000 system.
An important addition to the D-Plex set of features is the use of uniquemolecular identifiers (UMI) to each transcript incorporated in the library.
Given this new addition, it is now possible to exclude PCR duplicates froma set of reads, thus improving the transcript expression quantification.

6

D-PLEX mRNA-SEQ KIT MANUAL

Kit Materials
The D-Plex mRNA-seq kit (C05030033) includes the D-Plex Total RNAseq kit (C05030031) and the D-Plex mRNA Capture module (C05030032).
Table 1. Components of the D-Plex Total RNA-seq kit (C05030031)
Component

Cap color

Qty (24 rxns)

Storage

Fragmentation Buffer (FB)

Yellow

24 µl-20°C/-4°F

Dephosphorylation Reagent (DR)Yellow

7.2 µl

-20°C/-4°F

Dephosphorylation Buffer (DB)Yellow

48 µl-20°C/-4°F

Tailing Reagent (TR)

Red

12 µl

-20°C/-4°F

Tailing Buffer (TB)

Red

24 µl

-20°C/-4°F

Reverse Transcription Reagent (RTR)

Purple24 µl-20°C/-4°F

Reverse Transcription Buffer (RTB)

Purple120 µl

-20°C/-4°F

RT Primer H UDI (RTPH_UDI)

Purple24 µl-20°C/-4°F

RT Primer M UDI (RTPM_UDI)

Purple24 µl-20°C/-4°F

Template Switching Oligo UDI (TSO_UDI)

Purple48 µl-20°C/-4°F

PCR Master Mix (PCRMM)

Green

1200 µl-20°C/-4°F

Nuclease-free Water

Clear

2000 µl

Room T°-20°C/-4°F

Positive Control HBR (CTL+)Black

12 µl (1 ng/µl)

Long term storage:-80°C/-112°F

Table 2. Components of the mRNA Capture module (C05030032)
Component

Cap color

Qty (24 rxns)

Storage

Oligo d(T)25 Beads

Blue

480 µl+4°C

2X RNA Binding BufferBlue

7.2 ml+4°C

Wash BufferBlue

14.4 ml+4°C

Tris BufferBlue

1.65 ml+4°C

Nuclease-Free WaterBlue

1.2 ml+4°C

Storage
The components should be stored at temperatures indicated in Table 1
and Table 2.
7Required Materials Not Provided
IndexesSpecific D-Plex indexes were designed and validated to fit this technology
and are not included in this kit, providing you with total flexibility. They canbe bought separately according to your needs. Please choose the format
that suits you best among the compatible references to:Table 3. D-Plex Unique Dual Indexes Modules and their correspondingreferences
Unique Dual Indexes Modules

FormatReference

D-Plex Unique Dual Indexes Module – Set A

24 rxns

C05030021

D-Plex Unique Dual Indexes Module – Set B

24 rxns

C05030022

General equipment and reagents• Gloves to wear at all steps• Single channel pipettes and corresponding RNase-free filter tips:
10 µl, 20 µl, 200 µl, 1,000 µl• Crushed ice• RNase AWAY™ decontamination reagent (ThermoFisher
Scientific, 10328011)• Nuclease-free tubes: 0.2 ml, 1.5 ml• Table top centrifuge with strip rotor• Vortex agitator• Tube holder for 0.2 ml, 1.5 ml tubes• DiaMag 0.2 ml tube magnetic rack (Diagenode, B04000001)• DiaMag 1.5 ml tube magnetic rack (Diagenode, B04000003)• Thermal cycler

8

D-PLEX mRNA-SEQ KIT MANUALmRNA capture• DiaMag 0.2 ml tube magnetic rack (Diagenode, B04000001)• Absolute ethanol (VWR, 20821.310)• Agilent 2100 Bioanalyzer® (Agilent) and Agilent RNA Pico 6000
(Agilent, 5067-1513)
Post-PCR libraries clean-up• Agencourt® AMPure® XP Beads (Beckman Coulter, A63881)• Absolute ethanol (VWR, 20821.310)• DNase-free, RNase-free ultrapure water
DNA library size and yield estimation• Agilent 2100 Bioanalyzer® Agilent and High Sensitivity DNA Kit(Agilent, 5067-4626)• Qubit® Fluorometer (Thermo Fisher Scientific) and Qubit® dsDNA
HS Assay Kit (Thermo Fisher Scientific, Q32851)

9Remarks Before Starting
General recommendations• Read the complete manual before first time use.
• Decontaminate the working area as well as all the tools used toperform the library preparation with RNase AWAY™ reagent.
• Wear gloves at all steps to protect the RNA sample fromdegradation by contaminants and nucleases.
• All containers and storage areas must be free of contaminants
and nucleases.
• Add enzymes to reaction solutions last and thoroughly
incorporate them by pipetting up and down the solution several
times, and never vortex reagents mix.
• When using the protocol for the first time for a definite type ofsample, we strongly recommend using the positive HBR control
included in the kit.
• It is recommended to use good quality total RNA sample (RIN ≥ 7)
as starting material for mRNA capture.
• Unless otherwise stated, the lid of the thermal cycler used tocarry the reactions should be set at 105°C.
Designing your D-Plex RNA-seq experiment
The D-Plex mRNA-seq protocol incorporates mainly exonic coding mRNAs
but also some long non-coding RNAs bearing a poly(A) tail in the final
library for sequencing.
If you work with already purified or enriched RNAs and you do not intend to
perform an enrichment for mRNA transcripts, please use the D-Plex Total
RNA-seq kit (C05030031) and proceed directly with the library preparation
protocol.
If you are specifically interested in small non-coding RNA discovery andprofiling, we recommend using the D-Plex Small RNA-seq kit (C05030001).

10

D-PLEX mRNA-SEQ KIT MANUALTherefore, the choice of the D-Plex RNA-seq kit should be made based ona clear understanding of the scientific question in a given environment
before proceeding to RNA-seq library preparation as the choice of
methodology will strongly impact the end results.
Template
The D-Plex mRNA-seq kit has been developed for efficient mRNA capture
and library preparation from good quality total RNAs (RIN ≥ 7).
After mRNA capture, if the quantity of recovered RNAs is not limited,
we recommend to assess the RNA concentration, integrity and quality
using the Agilent RNA 6000 Pico kit (Agilent, 5067-1513) before library
preparation. This is only possible if the starting amount of total RNAsis high enough to recover sufficiently enriched RNAs which can then be
detected by the BioAnalyzer® system.
If quantification is not possible because of limited amount of material,
we recommend using a control sample at high concentration in parallel
to assess a percentage of recovery. This recovery rate (usually 0.1-1% formRNA capture) can be calculated and taken into account to estimate the
RNA quantity used for library preparation.
Table 4. Estimation of the quantity of recovered RNAs after mRNA capture
Total RNA starting amountCaptured mRNA expected amount

1 µg

1 – 10 ng

100 ng100 pg – 1 ng

50 ng

50 pg – 500 pg

10 ng

10 pg – 100 pg

11Figure 1: RNA electrophoregram of poly(A) selected mRNAs after the mRNA capture from 1 µg total Human
Brain RNA (HBR) using the D-Plex mRNA Capture module (Diagenode, C05030032).

12

D-PLEX mRNA-SEQ KIT MANUALStarting material
The D-Plex mRNA-seq protocol overall protocol, which includes first the
D-Plex mRNA capture protocol and then the D-Plex Total RNA-seq library
preparation protocol, has been validated for starting amounts ranging
from 10 ng up to 5 µg of purified total RNAs.
A starting volume of 50 μl is used in the initial mRNA capture process.
RNA templates should be dissolved in nuclease-free water in this volume
before starting the mRNA capture protocol.
The RNA sample should be free of salts (Mg2+, guanidinium salts) ororganic compounds (phenol, ethanol).
Multi-sample protocol
The protocol describes the library preparation process for one single
sample. If the interest is to prepare more than one sample in parallel,
please scale up accordingly.
Multiplexing advices
The PCR primers can be used for library multiplexing up to 48. In caseof a multiplexing scenario, it is recommended to follow Illumina’s librarypooling guidelines that are explained in the D-Plex Unique Dual Indexes
manual.
Positive control: total human brain RNA
A positive control is supplied in the kit for 12 reactions at a ready-to-use
concentration of 1 ng/μl. This control RNA is meant to monitor the library
preparation efficiency. Typically, 1 ng of control RNA prepared with the
D-Plex protocol and amplified during 14 PCR cycles will yield a minimumof 5 ng/µl of dsDNA after library purification in 20 µl.

13Figure 2: DNA electrophoregram of a D-Plex Total RNA-seq library prepared from 1 ng total Human Brain
RNA (HBR). 1 ng of the library was loaded on the Bioanalyzer.

14

D-PLEX mRNA-SEQ KIT MANUALSH OR T
PR OTOCO L
FOR EXPERIENCED USERS

Short Protocol For Experienced Users
Starting material: 10 ng - 5 µg of purified total RNAs in a 50 μl total volume of
nuclease-free water.

1.

Capture poly(A)-tailed RNAs using oligo(dT) magnetic beads. Pleaserefer to Part A of the detailed protocol for complete instructions.

2.

Transfer 7 µl of captured mRNAs in a RNase-free tube.

3.

Add 1 µl of Fragmentation Buffer (FB), incubate for 7 minutes at
94°C, and then cool down on ice.

4.

Prepare the Dephosphorylation Master Mix (DMM) by mixing 2 µlof Dephosphorylation Buffer (DB) and 0.3 µl of Dephosphorylation
Reagent (DR) in a RNase-free tube.

5.

Add 2 µL of the DMM to the sample, incubate for 15 minutes at 37°C,
and then cool down on ice.

6.

Prepare the Tailing Master Mix (TMM) by mixing 1 µl of Tailing Buffer(TB) and 0.5 µl of Tailing Reagent (TR) in a RNase-free tube.

7.

Add 1.5 µl of TMM to the sample, incubate for 40 minutes at 37°C +20 minutes at 65°C, and then cool down on ice.

8.

Add 1 µl of Reverse Transcription Primer UDI (RTP_UDI) to thesample on ice:
- For 50 ng - 500 pg captured mRNA, use RTPH_UDI- For 500 pg - 50 pg captured mRNA, use RTPM_UDI

16

9.

Incubate for 10 minutes at 70°C, then slowly decrease to 25°C by
ramping down at 0.5°C/second, and incubate for 2 minutes at 25°C.

10.

Take the Template Switch Oligo UDI (TSO_UDI) tube out of thefreezer and let it thaw on ice.

11.

Prepare the Reverse Transcription Master Mix (RTMM) by mixing
5 µl of Reverse Transcription Buffer (RTB) and 1 µl of Reverse

D-PLEX mRNA-SEQ KIT MANUAL

12.

Add 6 µl of RTMM to the sample and incubate for 15 minutes at 25°C.

13.

Add 2 µl of TSO_UDI to the sample and incubate for 120 minutes at
42°C + 10 minutes at 70°C.

14.

Add 20 µl of D-Plex Primer UDI and 50 µl of PCR Master Mix(PCRMM) to the sample.

15.

Proceed to PCR reaction and amplify the library according to thefollowing program:Temperature

Time & Cycles

98°C

30 seconds

98°C

15 seconds

72°C

60 seconds

72°C

SHORT PROTOCOLTranscription Reagent (RTR) in a RNase-free tube. Place the RTMM
on ice until addition to the sample.

n cycles*10 minutes

Hold at 4°C or freeze until further processing*Perform n cycles depending on the initial RNA input.See detailed protocol for
the PCR cycle table.

16.

Clean-up the PCR product with 72 µL of AMPure® XP beads (0.8X)
and elute the purified library in 50 μl of nuclease-free water.

17.

Perform a second clean-up with 40 µL of AMPure® XP beads (0.8X)
and elute the final library in 10-20 μl nuclease-free water (seedetailed protocol for more information).

18.

Perform library quantification using QuBit® dsDNA HS Assay Kit
and quality check using Bioanalyzer® DNA High Sensitivity Assay Kit
according to the manufacturer’s instructions.

17

DETA I L ED
PROTOCO L
PART A: D-Plex mRNA Capture
STEP 1- mRNA capture using oligo(dT) beads
PART B: D-Plex library preparation

19
19
23

STEP 2 - RNA fragmentation and dephosphorylation

23

STEP 3 - RNA tailing

25

STEP 4 - Reverse transcription with template switching

26

STEP 5 - PCR amplification

28

STEP 6 - Library clean-up

29

STEP 7 - Library QC and quantification

31

STEP 1
D-Plex mRNA Capture using
oligo(dT) beads - 1 hour

DETAILED PROTOCOLPART A: D-Plex mRNA Capture

Beads washing
1.1

Vortex and transfer 20 μl of Oligo d(T)25 Beads in a RNase-free
tube.

1.2

Add 100 µl of 2X RNA Binding Buffer.

1.3

Place the tube on a magnetic rack for 2 minutes to separate thebeads from the supernatant.

1.4

When the solution is clear, carefully remove and discard thesupernatant without disturbing the beads.

1.5

Repeat steps 1.2-1.4 once for a total of 2 washes.

1.6

Remove the tube from the magnetic rack.

1.7

Add 50 μl of 2X RNA Binding Buffer to resuspend the Oligo d(T)25
Beads.

First D-Plex mRNA Capture
1.8

Add 50 μl of total RNA sample to the 50 µl of Oligo d(T)25 Beads
from step 1.7.

1.9

Mix well by slowly pipetting the entire volume up and down at least
6 times.

19

1.10

Place the sample in a thermal cycler and run the following
program:
Temperature

Time

65°C

5 min.

4°C

5 min. hold

1.11

Remove the sample from the thermal cycler when the temperaturereaches +4°C.

1.12

Mix well by slowly pipetting the entire volume up and down at least
6 times.

1.13

Place the tube on a tube holder.

1.14

Incubate for 5 minutes at room temperature to allow thepoly(A)-tailed RNAs to bind to the beads.

1.15

Resuspend the beads by slowly pipetting the entire volume up anddown at least 6 times.

1.16

Place the tube on a tube holder.

1.17

Incubate for 5 minutes at room temperature.

First wash

20

1.18

Place the tube on the magnetic rack for 2 minutes to separate thebeads from the supernatant.

1.19

When the solution is clear, carefully remove and discard thesupernatant without disturbing the beads.

1.20

Remove the tube from the magnetic rack.

1.21

Add 200 µl of Wash Buffer to remove the unbound RNAs.

1.22

Mix well by slowly pipetting the entire volume up and down at least
6 times.

D-PLEX mRNA-SEQ KIT MANUAL

Place the tube on the magnetic rack for 2 minutes.

1.24

When the solution is clear, carefully remove and discard thesupernatant without disturbing the beads.

1.25

Repeat steps 1.21-1.24 once for a total of 2 washes.

1.26

Remove the tube from the magnetic rack.

Second mRNA capture
1.27

Add 50 μl of Tris Buffer.

1.28

Mix well by slowly pipetting the entire volume up and down at least
6 times.

1.29

Place the sample in a thermal cycler and run the following
program:
Temperature

Time

80°C

2 min.

25°C

5 min. hold

1.30

Remove the sample from the thermal cycler when the temperaturereaches +25°C.

1.31

Add 50 μl of 2X RNA Binding Buffer.

1.32

Mix well by slowly pipetting the entire volume up and down at least
6 times.

1.33

Place the tube on a tube holder.

1.34

Incubate for 5 minutes at room temperature to allow the poly(A)tailed RNAs to bind to the beads.

1.35

Resuspend the beads by slowly pipetting the entire volume up anddown at least 6 times.

1.36

Place the tube on a tube holder.

DETAILED PROTOCOL

1.23

21

1.37

Incubate for 5 minutes at room temperature.

Second wash
1.38

Place the tube on the magnetic rack for 2 minutes to separate thebeads from the supernatant.

1.39

When the solution is clear, carefully remove and discard thesupernatant without disturbing the beads.

1.40

Remove the tube from the magnetic rack.

1.41

Add 200 µl of Wash Buffer.

1.42

Mix well by slowly pipetting the entire volume up and down at least
6 times.

1.43

Place the tube on the magnetic rack for 2 minutes.

1.44

When the solution is clear, carefully remove and discard thesupernatant without disturbing the beads.

1.45

Remove the tube from the magnetic rack.

Elution22

1.46

Add 7 μl of Tris Buffer.

1.47

Place the sample in a thermal cycler and incubate for 2 minutes
at 80°C.

1.48

Spin down the sample briefly in a tabletop centrifuge andimmediately place the tube back on the magnetic rack to separate
the beads from the supernatant.

1.49

When the solution is clear, transfer the 7 μL of supernatantcontaining poly(A)-tailed RNAs to a clean RNase-free tube.

1.50

Place the sample on ice and proceed with D-Plex Total RNA-seq
library preparation or, alternatively, store the sample at -80°C
until further use.

D-PLEX mRNA-SEQ KIT MANUALSTEP 2RNA fragmentation anddephosphorylation - 30 minutes
2.1

Add 1 μl of Fragmentation Buffer (FB) to the captured mRNA
sample.

2.2

Mix by pipetting up and down at least 5 times.

2.3

Place the sample in a thermal cycler and incubate for 7 minutes
at 94°C to fragment the RNA.

2.4

Spin down the sample in a table top centrifuge and place on ice
until further processing.

2.5

Prepare a Dephosphorylation Master Mix (DMM) in a RNase-free
tube as follows:
Component

VolumeDephosphorylation Buffer (DB)2 μl

Dephosphorylation Reagent (DR)0.3 μlTotal Volume

DETAILED PROTOCOLPART B: D-PLEX LIBRARY PREPARATION

2.3 μl2.6

Mix by pipetting up and down at least 5 times and use immediately.

2.7

Add 2 μL of DMM to the sample. Unused DMM can be stored at-20°C and thawed up to 3 times.

23

24

2.8

Mix by pipetting up and down at least 5 times.

2.9

Incubate for 15 minutes at 37°C on thermal cycler.

2.10

Spin down the sample in a table top centrifuge and place on ice
until further processing.

D-PLEX mRNA-SEQ KIT MANUALRNA tailing 3.1

1 hourDETAILED PROTOCOLSTEP 3
Prepare a Tailing Master Mix (TMM) in a RNase-free tube as follows:
Component

VolumeTailing Buffer (TB)

1 µl

Tailing Reagent (TR)0.5 µlTotal Volume

1.5 µl3.2

Mix by pipetting up and down at least 5 times and use immediately.

3.3

Add 1.5 µl of TMM to the sample.

3.4

Mix by pipetting up and down at least 5 times.

3.5

Spin down the sample briefly (1-2 seconds) in a table top centrifuge.

3.6

Incubate for 40 minutes at 37°C + 20 minutes at 65°C on thermal
cycler, and then cool down the sample on ice for 2 minutes.

3.7

Spin down the sample in a table top centrifuge and place on ice until
further processing.

25

STEP 4
Reverse transcription
with template switching - > 2 hours
4.1

Add 1 µl of Reverse Transcription Primer UDI (RTP_UDI) to thesample on ice. Choose the right RTP according to your captured
mRNA amount (quantified or estimated after mRNA capture):
Captured mRNA amount26

50 pg – 500 pg

500 pg – 50 ng

RTPM_UDI

RTPH_UDI

4.2

Mix by pipetting up and down until the solution is homogenous.

4.3

Place the sample in a thermal cycler and run the following program:Temperature

Time

70°C

10 min70°C - 25°C

0.5°/sec

25°C

2 min hold

4.4

Spin down the sample in a table top centrifuge and place on ice until
further processing.

4.5

Take the Template Switch Oligo UDI (TSO_UDI) tube out of thefreezer and let it thaw on ice.

D-PLEX mRNA-SEQ KIT MANUALPrepare a Reverse Transcription Master mix (RTMM) in a RNasefree tube on ice as follows:
Component

VolumeReverse Transcription Buffer (RTB)

5 µl

Reverse Transcription Reagent (RTR)

1 µl

Total Volume

6 µl

4.7

Mix by pipetting up and down at least 5 times and use immediately.

4.8

Add 6 µl of RTMM to the sample.

4.9

Mix by pipetting up and down until the solution is homogenous.

DETAILED PROTOCOL

4.6

4.10 Incubate for 15 minutes at 25°C on thermal cycler and then place onice.
4.11 Add 2 µl of TSO_UDI to the sample. TSO_UDI should be stored at-20°C after use.
4.12 Mix by pipetting up and down until the solution is homogeneous.
Incubate for 120 minutes at 42°C + 10 minutes at 70°C on thermal cycler andthen cool down the sample on ice until further processing.
NOTE: The Reverse Transcription (120 minutes at 42°C + 10 minutes at 70°C)
may be performed overnight. If so, add an additional hold at +4°C once the two
steps are completed.

4.13 Spin down the sample in a table top centrifuge to collect the libraryat the bottom and proceed with next steps or store the sample at-20°C until further use.

27

STEP 5
PCR amplification -1 hour5.1

Add 20 µl of D-Plex Primer UDI (primer pair UDI#) to the sample.

5.2

Add 50 µl of PCR Master Mix (PCRMM) to the sample.

5.3

Mix by pipetting up and down until solution is homogeneous.

5.4

Determine the number of cycles (n) for PCR amplification according
to the captured mRNA amount (quantified or estimated after mRNA
capture:Starting amountn cycles (*)

50 ng

8

10 ng

10

5 ng

12

1 ng

14

100 pg

16

50 pg

17-18

* Depending on the RNA template, quantity and quality of the sample, 1 or 2 PCR
cycles can be added to generate more library (but in detriment of the duplicatescontent). The PCR amplification cycles are only provided here as guidelines
and should be adjusted according to your D-Plex RNA-seq experiment.

5.5

Incubate for PCR amplification according to the following program:
Step

Temperature

Time & Cycles

1. Initial denaturation

98°C

30 seconds

2. Denaturation

98°C

15 seconds

3. Annealing & extension

72°C

60 seconds

4. Final extension

72°C

10 minutes

Hold at 4°C or stored at -20°C until further processing

28

D-PLEX mRNA-SEQ KIT MANUAL

n cyclesLibrary clean-up
6.1

Take the AMPure® XP beads out of the fridge and resuspend them
gently on a rotating wheel or orbital shaker at room temperature
before use.

6.2

Add 72 µL of AMPure® XP beads (0.8X) to the 90 µL of the amplified
DNA library from section 5.

6.3

Mix thoroughly by pipetting up and down several times or by vortexinga few seconds.

6.4

Incubate for 5 minutes at room temperature under mild agitation.

6.5

Spin down the sample in a table top centrifuge and place the tube
on a magnetic rack for 3 minutes to separate the beads from thesupernatant.

6.6

When the solution is clear, carefully remove and discard thesupernatant without disturbing the beads.

6.7

While keeping the tube on the magnetic rack, add 200 µL of freshly
prepared 80% ethanol for 30 seconds to wash the beads.

6.8

Carefully remove and discard the supernatant without disturbing thebeads.

6.9

Repeat steps 6.7-6.8 once for a total of 2 washes.

DETAILED PROTOCOLSTEP 66.10 Spin down the sample in a table top centrifuge, place the tube backon the magnetic rack and remove any remaining ethanol.
6.11 Leave the tube open for 2-3 minutes to let the beads pellet dry (do29not over-dry the beads as it may result in low recovery, over-driedbeads become light brown with cracks) and then remove it from themagnetic rack.
6.12 Add 50 µL of nuclease-free water.
6.13 Mix slowly by pipetting up and down to resuspend the beads.
6.14 Incubate the beads in water for 2 minutes at room temperature
under mild agitation.
6.15 Spin down the sample in a table top centrifuge and place the tube
on the magnetic rack for 3 minutes to separate the beads from thesupernatant.
6.16 When the solution is clear, carefully collect the 50 µL of supernatant
without taking up any beads.
6.17 Transfer the cleaned-up library in a RNase-free tube.
6.18 Repeat the clean-up procedure (from steps 6.2 to 6.17) with 40 µL
AMPure® XP beads (0.8X) but elute the purified library in 20 µL of
nuclease-free water. If more concentrated library is needed, theelution can be done in 10 µl of nuclease-free water.
6.19 Store the final cleaned-up library at -20°C until further use.

30

D-PLEX mRNA-SEQ KIT MANUALLibrary QC and quantification
7.1

For quantification, measure the library concentration using QuBit®
dsDNA HS Assay kit according to the manufacturer’s instructions.

7.2

For library size estimation, use the Bioanalyzer® DNA High Sensitivity
assay kit according to the manufacturer’s instructions.

DETAILED PROTOCOLSTEP 731

D-Plex mRNA Construct

Figure 3: The D-Plex mRNA-seq UDI library construct bears the Truseq (Illumina) HT adapters with uniquedual indexes (UDI) and unique molecular identifiers (UMI). Read 1 = UMI – TSM– Insert – A-tail – 3’ adapter;
Read 2 = Insert – TSM – UMI.

32

D-PLEX mRNA-SEQ KIT MANUALSequencing Recommendations
The D-Plex mRNA-seq library construct bears the TruSeq (Illumina) HT
adapters with unique dual indexes (UDI). In case of a multiplexing scenario,
it is therefore recommended to submit the D-Plex libraries as TruSeq HTlibraries to your sequencing provider.
The complete architecture of the D-Plex mRNA-seq construct is provided
in Figure 3. The empty library size (adapters + template switch + UMI + Atail) is equal to 172bp.
Given the high complexity of the coding transcriptome in D-Plex mRNAseq libraries, it is advised to sequence one library with a minimum of
20-25 million reads in order to sufficiently cover the library content for
downstream analysis.
It is recommended to perform the sequencing of the D-Plex libraries
in single-end mode as the quality of the sequencing data tends to bebetter than in paired-end mode. Furthermore, if the interest is mainly
gene expression analysis, the current sequencing configurations such asin Illumina’s NovaSeq system allow a sufficient (if not total) coverage ofthe insert by the read length in single end.
The read 1 sequence starts with the UMI and the template switch motif(for a total of 16 bases) that will be removed during the downstream
bioinformatics analysis. Therefore, we recommend a read length of 100
bp sequences for mRNA-seq library.
Example: 1 x 100 bp Single-end sequencing will give (after trimming) a
Read 1 sequence length = 84 bp.

33

However, if paired-end sequencing is needed, we recommend using ourCustom Sequencing Primer for Read2 (CSPR2) that has to be orderedseparately (C05020001). This custom sequencing primer can be spiked inwith the standard Illumina primer for read2. If more information is neededon setting up a paired-end sequencing with the D-Plex libraries, pleasecontact us (customer.support@diagenode.com).

34

D-PLEX mRNA-SEQ KIT MANUAL

Data Analysis Recommendations
The D-Plex libraries contain special sequences that need particulartreatment in order to get the best results out of your datasets. The
D-Plex construct holds special sequences namely the UMI, the A-tail,and the template switch motif. This guide will take you through the basic
processes of trimming, alignment and counting, complemented with
an optional UMI processing, using software tools and settings that we
validated. Though naturally other tools and methods can also be used,
please pay attention to finding the optimal settings for your experiments.
In our example commands, we assume that the necessary software tools
have been downloaded from the links provided at the end of the section
and are in the PATH.
We recommend the fumi-tools software package for UMI processing. If
the UMIs are not of interest, their processing can safely be skipped andthe rest of the pipeline will not change. In such a case, the UMI sequences
will be only removed from the reads during trimming and will be ignored
for the rest of the analysis.
Optional
UMI
PREPROCESSING

Main pipeline
TRIMMING

ALIGNMENT

Optional
DEDUPLICATIONBASED ON UMIs

Main pipeline
COUNTING

Figure 6: D-Plex Total RNA-seq bioinformatics pipeline

UMI-preprocessing (optional)
To process the UMIs, the first thing to do is to copy the UMI sequence tothe read ID. The first 12 bases in the read (from the 5’ end) correspond to
the UMI sequence. In the fumi-tools package the copy_umi command cancopy these bases to the correct lines in the fastq file. The tool expects afastq file as input, which can be gzip compressed, and it will output a fastq
file, which will be either gzip compressed or uncompressed, based on
the extension set on the commandline (.gz for compression). Besides the
input, the command needs one mandatory option: the length of the UMI

35

(to copy from the read to the ID line) as input. Optionally, the computationaltime can be reduced by increasing the number of CPUs to be used via thethreads parameter. The example command below uses all the 12 UMI
bases (recommended) and 10 threads.

Single-end processing
fumi_tools copy_umi --threads 10 --umi-length 12 -i
reads.fastq.gz -o reads_w_umi.fastq.gz

Paired-end processing
fumi_tools copy_umi --threads 10 --umi-length 12 -i
reads_R1.fastq.gz --input-read2 reads_R2.fastq.gz -oreads_R1_w_umi.fastq.gz --output-read2 reads_R2_w_
umi.fastq.gz
Trimming
Trimming is mandatory for the reads generated with the D-Plex RNA-seq
kit. The aim of trimming is to remove these artificial sequences typical of
D-Plex construct (UMI, A-tail and template switch motif) that will likely
hamper downstream analyses.
In our example command below, we use cutadapt package to properly
trim the reads and obtain a read set as clean as possible. The commands
remove UMI, A-tail/T-tail, template switch motif and adapters sequencesfrom all reads and discard reads which result shorter than 15 bases (these
are considered too short to be included in the analysis.). The trimming
commands can be applied on the input raw files (reads.fastq.gz) coming
directly from the sequencer or alternatively, on the UMI pre-processed
files (reads_w_umi.fastq.gz) if the UMIs are of interest.

36

D-PLEX mRNA-SEQ KIT MANUALSingle-end processing
cutadapt --trim-n --match-read-wildcards -u 16-n 4 –a AGATCGGAAGAGCACACGTCTG -a AAAAAAAA -a
GAACTCCAGTCAC -e 0.2 --nextseq-trim 20 -m 15 –otrimmed_reads.fastq.gz raw_reads_w_umi.fastq.gz

Paired-end processing
cutadapt --trim-n --match-read-wildcards -u
16 -n 4 -a "AGATCGGAAGAGCACACGTCTG;e=0.2" -a"AAAAAAAA;e=0.2" -a "GAACTCCAGTCAC;e=0.2"' -A"CCCNNNNNNNNNNNNNAGATC;o=21" -G "TTTTT" --nextseqtrim 20 -m 15:15 –o trimmed_reads.fastq.gz raw_
reads_R1.fastq.gz -p trimmed_reads_R2.fastq.gz raw_
reads_R1_w_umi.fastq.gz raw_reads_R2_w_umi.fastq.gz
Alignment
Aligning the trimmed reads needs no special treatment as you can use anyaligner that is suitable for mapping RNA-seq reads. First and foremost,
we recommend aligning to the genome (instead of the transcriptome).
Indeed, D-Plex tends to generate very high-complexity libraries which
often include RNAs that are not identified yet, and therefore, would not
map to a transcriptome reference consisting of only known transcripts.
Of course, in addition to the genome alignment, the mapped reads canbe assigned to known transcripts as well for expression analysis of the
known genes.
We recommend using the STAR alignment software. To run STAR, you needto provide the following arguments to the program: the input trimmed
reads data file; the length at which reads were sequenced minus 1; thefolder containing the STAR indexed genome (see STAR user guide formore information on how to prepare the genome); optionally the numberof CPUs to be used and the output format.
The example command below shows how to run STAR on the trimmed
reads data. We assume we are dealing with a human sample, that reads
were sequenced at 50 bases long and that we want to use 10 CPUs.
37

Please adapt the read length if it is longer than 50 bp and change the hg19
genome if your samples are not human.

Single-end processing
STAR --runThreadN 10 --readFilesCommand zcat--genomeDir /genomes/hg19/ --sjdbGTFfile /genomes/
hg19/hg19.gtf --sjdbOverhang 49 --readFilesIntrimmed_reads.fastq.gz--quantMode TranscriptomeSAM--quantTranscriptomeBAMcompression -1 --outSAMtype
BAM SortedByCoordinate --outSAMunmapped Within--outFileNamePrefix ./MySample_

Paired-end processing
STAR --runThreadN 10 --readFilesCommand zcat--genomeDir /genomes/hg19/ --sjdbGTFfile /genomes/
hg19/hg19.gtf --sjdbOverhang 49 --readFilesIntrimmed_reads_R1.fastq.gz trimmed_reads_R2.fastq.gz--quantMode TranscriptomeSAM--quantTranscriptomeBAMcompression -1 --outSAMtype
BAM SortedByCoordinate --outSAMunmapped Within--outFileNamePrefix ./MySample_
Deduplication based on UMIs (optional)
UMI processing enables the distinction between identical reads that arecoming from different RNA molecules and identical reads resulting from
PCR amplification. (If UMI deduplication is chosen, notice that you shouldhave applied the UMI pre-processing step before trimming). Below weprovide the command example to use fumi-tools to remove PCR clones
from the alignment files. Computational resources to be used may bechanged as desired using threads parameter (number of CPUs) andmemory.

38

D-PLEX mRNA-SEQ KIT MANUALTo remove duplicates from the genome alignment, no sorting is needed
before UMI deduplication. However, to remove duplicates from thetranscriptome alignment output, since the alignments are not sorted,
please use samtools command provided below before UMI deduplication:
samtools sort -@ 10 -o MySample_Aligned.
toTranscriptome.sorted.out.bam MySample_Aligned.
toTranscriptome.out.bam
Now you can deduplicate both the genome and the transcriptome alignment
with the UMI deduplication command of fumi-tools called dedup:
fumi_tools dedup --threads 10 --memory 10G -i
MySample_Aligned.toTranscriptome.sorted.out.bam -o
MySample_deduplicated_transcriptome.bam
fumi_tools dedup --threads 10 --memory 10G -i
MySample_Aligned.sortedByCoord.out.bam -o MySample_
deduplicated_genome.bam
Note that fumi-tools outputs name-sorted bam files, these are ready to
be used as input for counting software tools. For another application, you
may want to sort the bam files differently (e.g. soring by coordinates).
Counting
The counting, or expression level calculation is the last step of theprocessing to generate an expression level matrix.
When studying the coding part of the transcriptome (exons only), counting
tools such as featureCounts or HTSeq-count may be used for counting
exon features and summarizing expression at gene level. Alternatively,
RSEM may be used to estimate expression at isoform level.
Notice that D-Plex produces forward-stranded data. Stranded libraries
have the benefit that reads map to the genome strand where they wereoriginated from. Therefore, when estimating transcript expression, readsaligned to the forward strand should be assigned only to transcript featuresin the forward strand whereas reads aligned to the reverse strand should
be assigned only to transcript features in the reverse strand. For this,make sure you select “stranded-mode” in any tool of choice.
39

Links for the tools used in the example pipeline
Tool

40

Link

Websitehttps://www.diagenode.com/en

Fumi-tools

https://ccb-gitlab.cs.uni-saarland.de/tobias/fumi_tools/releases

Cutadapt

https://github.com/marcelm/cutadapt

STAR

https://github.com/alexdobin/STAR

Samtools

http://www.htslib.org/download

RSEM

http://deweylab.github.io/RSEM

featureCounts

http://subread.sourceforge.net

Htseqcounthttps://htseq.readthedocs.io/en/master/count.html

D-PLEX mRNA-SEQ KIT MANUALExample of Results
D-Plex mRNA-seq UDI library construct

Figure 4: DNA electropherogram of a D-Plex mRNA-seq UDI library construction made from 1 µg of Human
Universal Reference RNA before mRNA capture. 1 ng of the library was loaded on the Bioanalyzer.

Figure 5: DNA electropherogram of a D-Plex mRNA-seq UDI library construction made from 10 ng of Human
Universal Reference RNA before mRNA capture. 1 ng of the library was loaded on the Bioanalyzer.

41

Related Products
Product

42

Reference

D-Plex Total RNA-seq Kit

C05030031

D-Plex Small RNA-seq Kit

C05030001

D-Plex Unique Dual Indexes Module – Set A

C05030021

D-Plex Unique Dual Indexes Module – Set B

C05030022

D-Plex mRNA Capture Module

C05030032

DiaMag 0.2 mL tube magnetic rack

B04000001

DiaMag 1.5 mL tube magnetic rack

B04000003

D-PLEX mRNA-SEQ KIT MANUALFOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
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