D-Plex Small RNA-seq Kit
Small RNA library preparation kit
for Illumina® sequencing
Cat. No. C05030001 (Core module - 24 rxns)

USER GUIDEV2 03_2024

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.

Get the electronic version of thisuser manual on the product pagewww.diagenode.com/en/p/D-Plex-Small-RNA-seq-Library-Prep-x24

Summary
Kit Method Overview

4

Introduction

5

Kit Materials

7

Required Materials Not Provided

8

Remarks Before Starting

10

Short Protocol (for experienced users)

13

Detailed Protocol

17

D-Plex Small RNA Construct

30

Sequencing Recommendations

32

Data Analysis Recommendations

34

Example of Results

40

Related Products

42

Revision History

42

Kit Method Overview
Time Pause

RNA

STEP
1

RNA

STEP
2

AAAAA...

RNA
STEP
3

UMI
+
5’ adaptor

rXrXrXrXRNA sample
preparation

5 min

RNA tailing45 min

Reverse transcription
with template switching>2h

AAAAAAA...V TTTTTTTT...XXXXAAAAAAA
TTTTTTTT

XXXX
XXXX

AAAAAAA
TTTTTTTTPCR forward
PCR reverse

STEP
4

AAAAAAA
TTTTTTTT

XXXX
XXXX

PCR amplification

DNA

4

STEP
5

Library purification

STEP
6

Library clean-up
and shaping

STEP
7

Library quality control
and quantification

D-PLEX SMALL RNA-SEQ KIT MANUAL

45 minIntroduction
The Diagenode D-Plex Small RNA-seq Library Preparation Kit is a tool
designed for the study of the small non-coding transcriptome. The
present kit incorporates the unique D-Plex technology to generate small
RNA libraries for Illumina sequencing.
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• Higher library complexity than most of other available library
preparation kits for small RNA-sequencing
The library preparation protocol works on either total intact RNAs (RIN≥8)
extracted and purified from a given sample or a small RNA fraction (<200nt),
that might very well represent the circulating content of a liquid biopsytype of sample (blood serum and plasma). The input requirements of themethod are flexible and allow the user to perform the method within awide range of RNA quantities going from 10 pg of small RNAs (< 200 nt)
or circulating RNAs up to 100 ng of total RNAs.
The core of the technology relies on ligation-free reactions to attach theIllumina adaptors to both ends of the library construct. Therefore, theresults generated with the D-Plex Small RNA-seq kit will vastly differ
from those produced with ligase-based approach. For instance, theresults generated with the D-Plex kit will encompass a vast spectrum ofsmall non-coding RNAs (miRNAs, snoRNAs, snRNAs, piRNAs) whereasa ligase-based approach will enrich the sequencing library in 5’-P – 3’OH RNAs, mainly mature miRNAs.
Diagenode therefore recommends having a clear understanding of thescientific question being asked in a given experiment before proceeding
to a small RNA-seq library preparation as the choice of technology willstrongly impact the end result.

5

For optimal workflow flexibility, the library preparation is available inboth unique dual index (UDI) and single index (SI) configurations. The
D-Plex Unique Dual Indexes Modules (C05030021, C05030022, C05030023
and C05030024) and the D-Plex Single Indexes Modules (C05030010 and
C05030011) are available separately from the library preparation kit,providing PCR primers for library multiplexing up to 96 (using the UDIsets). The use of UDI is highly recommended to mitigate errors introduced
by read misassignment, including index hopping frequently observed withpatterned flow cells such as Illumina’s NovaSeq 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 SMALL RNA-SEQ KIT MANUAL

Kit Materials
Table 1: Components of the D-Plex Small RNA-seq kit (C05030001)
Component

Cap color

Qty (24 rxns)

Storage

Dephosphorylation Buffer (DB)Yellow

48 µL-20°C/-4°F

Dephosphorylation Reagent (DR)Yellow

12 µL-20°C/-4°F

Crowding Buffer (CB)Yellow

120 µL+4°C/+40°F

Small Tailing Reagent (STR)

Red

12 µL

-20°C/-4°F

Small Tailing Buffer (STB)

Red

24 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

Reverse Transcription Reagent (RTR)

Purple24 µL-20°C/-4°F

Reverse Transcription Buffer (RTB)

Purple120 µL-20°C/-4°F

Small Template Switching Oligo UDI(STSO_UDI)

Purple

48 µL-20°C/-4°F

PCR Master Mix (PCRMM)

Green

1200 µL-20°C/-4°F

Nuclease-free Water

Clear

2000 µL

20°C/-4°F-20°C/-4°F

Positive Control miRNA (CTL+)

Black

12 µL (1 ng/µL)

Long term storage:-80°C/-112°F

Important Notice
The RT Primer H, RT Primer M and Small Template Switching Oligo
components included in the D-Plex Small RNA-seq kit (C05030001) areonly suitable for UDI library construction. The tubes are labelled witha “UDI” tag. For SI library construction, the RT Primer H, RT Primer M
and Small Template Switching Oligo components suitable for SI library
construction are included in the D-Plex Single Indexes modules (C05030010
and C05030011). You should use the components corresponding to thedesired – UDI or SI – library construction.
Storage
The components should be stored at temperatures indicated in Table 1.

7Required Materials Not Provided
IndexesSpecific D-Plex indexes were designed and validated to fit this technology
and are not included in this core module, providing you with total flexibility.
They can be bought separately according to your needs. Please choose
the format that suits you best among the compatible references to.
Table 2: D-Plex Indexes Modules and their corresponding references
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

D-Plex Unique Dual Indexes Module – Set C

24 rxns

C05030023

D-Plex Unique Dual Indexes Module – Set D

24 rxns

C05030024

Single Indexes Modules

FormatReference

D-Plex Single Indexes Module – Set A

24 rxns

C05030010

D-Plex Single Indexes Module – Set B

24 rxns

C05030011

For complete workflow, the D-Plex Small RNA-seq kit (C05030001) should
be ordered together with D-Plex Indexes modules (Table 2). The final
D-Plex library construction will either bear UDI or SI (Table 3).
Table 3: D-Plex library construction
D-Plex UDI Library Construction
D-Plex Small RNA-seq Kit

C05030001

AND

D-Plex Unique Dual Indexes Module

8

D-PLEX SMALL RNA-SEQ KIT MANUAL

D-Plex SI Library Construction
D-Plex Small RNA-seq Kit

C05030001

AND
C05030021
C05030022
C05030023
C05030024

D-Plex Single Indexes Module

C05030010
C05030011

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• RNase AWAY™ decontamination reagent (Thermo Fisher
Scientific, 10328011)• Cruched ice• RNase-free tubes: 0.2 ml, 0.5 ml, 1.5 ml• Table top centrifuge with strip rotor• Vortex agitator• Tube holder for 0.2 ml, 0.5 ml, 1.5 ml tubes• DiaMag 0.2 ml tube magnetic rack (Diagenode, B04000001)• Thermal cycler
Post-PCR libraries purification• MicroChIP DiaPure kit (Diagenode, C03040001)• DNase-free, RNase-free ultrapure water
Post-PCR libraries clean-up• Agencourt® AMPure® XP Beads (Beckman Coulter, A63881)• Absolute ethanol (VWR, 20821.310)• DNase-free, RNase-free ultrapure water
Post-PCR libraries size selection
Agarose (4-6%) or polyacrylamide (4-6%) self-made/pre-casted gels with
the necessary electrophoresis equipment (TBE 0.5X buffer, electrophoresis
tank, generator…) including the material required for the gel cut (scalpel
blade, tweezers and pestle) and the supplementary MinElute® Gel
Extraction Kit (Qiagen, 28604).
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.
• The D-Plex Small RNA-seq protocol is essentially the same forboth UDI and SI library constructions. However, the RT Primer H,
RT Primer M and Small Template Switching Oligo are specific tothe desired – UDI or SI – library construction. The RT Primer H,
RT Primer M and Small Template Switching Oligo components
included in the D-Plex Small RNA-seq kit (C05030001) are only
suitable for UDI library construction. The tubes are labelled with a“UDI” tag. For SI library construction, the RT Primer H, RT Primer
M and Small Template Switching Oligo components only suitablefor SI library construction are included in the D-Plex Single Indexes
Modules (C05030010 and C05030011). You should carefully use thecomponents corresponding to the desired – UDI or SI – library
construction.
• 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 from degradationby contaminants and nucleases.
• All containers and storage areas must be free of contaminants andnucleases.
• Add enzymes to reaction solutions last and thoroughly incorporate
them by pipetting up and down the solution several times.
• When using the protocol for the first time for a definite type ofsample, we strongly recommend using the positive miRNA control
included in the kit.
• Unless otherwise stated, the lid of the thermal cycler used to carry
the reactions should be set at 105°C.
Template
Both total RNA and small RNA (<200 nt) templates can be used as input
for the D-Plex Small RNA-seq library preparation.
10

D-PLEX SMALL RNA-SEQ KIT MANUALSmall RNAs can be isolated either from biological fluids (e.g. bloodplasma or serum), exosomes or from cells using a column-based method(e.g. miRNeasy Mini Kit (Qiagen, 217004)) capable of retaining the small
RNA fraction (< 200 nt). Please be aware that not all extraction kits on themarket are able to efficiently isolate RNAs under 200 nt.
The D-Plex Small RNA-seq kit has also been developed with high-quality
total RNAs (RIN > 8), but the kit can also perform well on partially and evenhighly degraded samples (e.g. FFPE-derived samples). However, sample
degradation may result in the underrepresentation of the small noncoding RNAs in the final library. For total RNA templates, we recommend
to assess the RNA integrity and quality using the Agilent RNA 6000 Picokit (Agilent, 5067-1531) before library preparation.
The D-Plex method offers an alternative protocol in which the RNA
sample is treated with a dephosphorylation reagent (DR), that will
dephosphorylate the 3’end of an RNA molecule (if it is phosphorylated) andmonophosphorylate the 5’end (if it is hydroxylated). This protocol might
constitute a special interest for researchers studying the degradation
products of coding RNAs and long non-coding RNAs.
Starting material
The D-Plex Small RNA-seq protocol has been validated for starting
amounts ranging from 10 pg to 10 ng for small RNAs (< 200 nt) or
circulating RNAs and ranging from 100 pg to 100 ng for total RNAs
(RIN ≥ 8) (Table 4).
Table 4: Input recommendations
Template

Starting amount

RIN

Total RNA

100 pg - 100 ng≥8

Small RNA (< 200 nt)

10 pg - 10 ng

N/A

Circulating RNA

10 pg - 10 ng

N/A

A starting volume of 8 μl is used in the library preparation process. RNA
templates should be dissolved in nuclease-free water in this volume
before starting the protocol.

11

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
and the D-Plex Single Indexes manuals.
Positive control: cel-miR-39-3p
A positive control is supplied in the kit for 12 reactions at a ready-to-use
concentration of 1 ng/µL. This synthetic miRNA is meant to be used in alibrary preparation to monitor the library preparation efficiency or as a
spike-in in a given RNA sample to account for an eventual matrix effect.
Typically, 1 ng of synthetic miR39 prepared with the D-Plex protocol andamplified during 12 PCR cycles will yield a minimum of 400 ng of dsDNA
after DiaPure purification.
The synthetic miRNA mimics the mature sequence of cel-miR-39-3p from
Caenorhabditis elegans and is 22 nt long.
5’-UCACCGGGUGUAAAUCAGCUUG-3’

Figure 2: DNA electrophoregram of a D-Plex Small RNA-seq SI library construction prepared from 1 ng of miR39(12X PCR cycles) after DiaPure purification. 1 ng of the library was loaded on the Bioanalyzer.

12

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

Short Protocol For Experienced Users
Notice before starting•

The D-Plex Small RNA-seq protocol is essentially the same for both UDI and SI library constructions.
However, the RT Primer H, RT Primer M and Small Template Switching Oligo are specific to the desired– UDI or SI – library construction. The RT Primer H, RT Primer M and Small Template Switching Oligo
components included in the D-Plex Small RNA-seq kit (C05030001) are only suitable for UDI library
construction. The tubes are labelled with a “UDI” tag. For SI library construction, the RT Primer H, RT
Primer M and Small Template Switching Oligo components only suitable for SI library construction are
included in the D-Plex Single Indexes modules (C05030010 and C05030011). You should carefully use
the components corresponding to the desired – UDI or SI – library construction.

•

The D-Plex Small RNA-seq library preparation incorporates the different classes of small non-coding
RNAs in the final library for sequencing. If coding RNA or long non-coding RNA degradation products
are of particular interest, we recommend to use the Dephosphorylation Reagent (DR) in step 3 toenrich the final library with those products.

1.

Take the Crowding Buffer (CB) out of the freezer and let it warm up
at room temperature for 30 minutes before using it at step 2. Fromthen on, CB can be stored at +4°C.

2.

Add 2 µl of Dephosphorylation Buffer (DB) and 5 µl of CB to the 8 µlof RNA sample.

3.

OPTIONAL [To proceed to RNA 3’-end dephosphorylation, add 0.5µl of Dephosphorylation Reagent (DR). Incubate for 15 minutes at
37°C and then place the sample on ice.]4.

Prepare the Small Tailing Master Mix (STMM) by mixing 1 µl of Small
Tailing Buffer (STB) and 0.5 µl of Small Tailing Reagent (STR) in a
RNase-free tube.

5.

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

6.

Add 1 µl of Reverse Transcription Primer (RTP) to the sample
on ice:• For 100 ng - 10 ng total RNA or 10 ng - 1 ng small RNA fraction
use RTPH.
• For 10 ng - 100 pg total RNA or 1 ng - 10 pg small RNA fraction
use RTPM.

14

D-PLEX SMALL RNA-SEQ KIT MANUAL

7.

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

8.

Take the Small Template Switch Oligo (STSO) tube out of the freezer
and let it thaw on ice.
CAUTION: Carefully use STSO corresponding to the desired - UDI or SI - library
construction.

9.

Prepare the Reverse Transcription Master Mix (RTMM) by mixing
5 µl of Reverse Transcription Buffer (RTB) and 1 µl of Reverse
Transcription Reagent (RTR) in a RNase-free tube. Place the RTMM
on ice until addition to the sample.

10.

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

11.

Add 2 µl of STSO to the sample.

SHORT PROTOCOLCAUTION: Carefully use RTPH and RTPM corresponding to the desired - UDI
or SI - library construction.

NOTE: Dilute STSO 1/5 if working on very low input samples; refer to detailed
protocol for more information.

12.

Incubate for 120 minutes at 42°C + 10 minutes at 70°C.

13.

For UDI library construction: Add 20 µl of D-Plex Primer UDI and 50µl of PCR Master Mix (PCRMM) to the sample.
For SI library construction: Add 10 µl of D-Plex Formard Primer, 10µl of indexed D-Plex Reverse Primer, and 50 µl of PCR Master Mix(PCRMM) to the sample.

14.

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

Time & Cycles

98°C

30 seconds

98°C

15 seconds

72°C

60 seconds

72°C

n cycles*10 minutes

Hold at 4°C or freeze until further processing

15

For SI library construction:
Temperature

Time & Cycles

98°C

30 seconds

98°C

15 seconds

62°C

30 seconds

72°C

30 seconds

72°C

n cycles*10 minutes
Hold at 4°C or freeze until further processing*Perform n cycles depending on the initial RNA input:- from 10 cycles: for 100 ng total RNAs or 10 ng small RNAs
- up to 20 cycles: for 100 pg total RNAs or 10 pg small RNAs

NOTE: If you plan to proceed to the size-selection of the library for short inserts(< 50 nt) (see sections 6 of the detailed protocol), add 2 more cycles to the ncycles displayed above.

16

15.

Purify the PCR product using the MicroChIP DiaPure kit (DiagenodeC03040001) according to the kit's instructions (follow the “Protocol
for ChIP samples”). To perform further clean-up or size-selection of
the library, refer to section 6 of the detailed protocol.

16.

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.

D-PLEX SMALL RNA-SEQ KIT MANUALDETA I L ED
PROTOCO L
STEP 1 - RNA sample preparation

20

STEP 2 - RNA tailing

21

STEP 3 - Reverse transcription with template switching

22

STEP 4 - PCR amplification

24

STEP 5 - DNA library purification

26

STEP 6 - DNA library clean-up and shaping

27

STEP 7 - Library QC and quantification

31

STEP 1
RNA sample preparation -18

5 minutes

1.1

Take the Crowding Buffer (CB) out of the freezer and let it warm up
at room temperature for 30 minutes before using it at step 1.2. Fromthen on, CB can be stored at +4°C.

1.2

Add 2 µl of Dephosphorylation Buffer (DB) and 5 µl of CB to the 8 µlof RNA sample. Pipet slowly as CB is viscous.

1.3

Mix by pipetting up and down until solution is homogenous.

D-PLEX SMALL RNA-SEQ KIT MANUALRNA tailing -45 minutes

2.1

OPTIONAL [To proceed to the RNA 3’-end dephosphorylation, add
0.5 µl of Dephosphorylation Reagent (DR) to the sample. Mix bypipetting up and down until solution is homogeneous. Incubate for
15 minutes at 37°C and then place the sample on ice.]

2.2

Prepare a Small Tailing Master Mix (STMM) in a RNase-free tube
according as follows:
Component

DETAILED PROTOCOLSTEP 2

VolumeSmall Tailing Buffer (STB)

1 µl

Small Tailing Reagent (STR)

0.5 µlTotal Volume

1.5 µl2.3

Mix by pipetting up and down and use immediately.

2.4

Add 1.5 µl of STMM to the sample.

2.5

Mix by pipetting up and down until solution is homogeneous.

2.6

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

2.7

Incubate for 40 minutes at 37°C on thermal cycler and then cool
down on ice for 2 minutes.

2.8

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

19

STEP 3
Reverse transcription with templateswitching - > 2 hours
CAUTION: Carefully use the RT Primer H, RT Primer M and Small Template
Switching Oligo components corresponding to the desired - UDI or SI – libray
construction:- The components suitable for the UDI library construction are included in
the D-Plex Small RNA-seq Kit (C05030001). The tubes are labelled with a“UDI” tag.
- The components suitable for the SI library construction are included in
the D-Plex Single Indexes Modules (C05030010 and C05030011).

3.1

Add 1 µL of Reverse Transcription Primer (RTP) to the sample on
ice. Choose the right RTP according to your starting RNA input:
Total RNA
100 pg – 10 ng

10 ng – 100 ng

10 pg – 1 ng

1 ng – 10 ng

RTPM

RTPH

RTPM

RTPH

3.2

Mix by pipetting up and down until solution is homogeneous.

3.3

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

3.4

20

Small RNA / Circulating RNA

Temperature

Time

70°C

10 min70°C - 25°C

0.5°/sec

25°C

2 min hold

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

D-PLEX SMALL RNA-SEQ KIT MANUALTake the Small Template Switch Oligo (STSO) tube out of the freezer
and let it thaw on ice.

3.6

Prepare a Reverse Transcription Master Mix (RTMM) in a RNasefree tube according as follows:
Component

VolumeReverse Transcription Buffer (RTB)

5 µl

Reverse Transcription Reagent (RTR)

1 µl

Total Volume

DETAILED PROTOCOL

3.5

6 µl

3.7

Mix by pipetting up and down and use immediately.

3.8

Add 6 µl of RTMM to the sample.

3.9

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

3.10 Incubate for 15 minutes at 25°C and then place the sample on ice.
3.11 Add 2 µl of STSO to the sample. STSO should be stored at -20°C
after use.
NOTE: In case of ultra-low input sample processing, please dilute the TSO 1/5:
- for total RNAs < 200 pg- for small RNAs and circulating RNAs < 50 pg

3.12 Mix by pipetting up and down until the solution is homogeneous.
3.13 Incubate for 120 minutes at 42°C + 10 minutes at 70°C, and thencool 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 additional hold at +4°C once the firsttwo steps are completed.

3.14 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.

21

STEP 4
PCR amplification 4.1

1 hourFor UDI Library construction: Add 20 µl of D-Plex Primer UDI(Primer UDI#) to the sample.
For SI library construction: Add 10 µl of D-Plex Forward Primer
and 10 µl of indexed D-Plex Reverser Primer to the sample.

4.2

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

4.3

Mix by pipetting up and down until solution is homogeneous.

4.4

Determine the number of cycles (n) you have to perform according
to the RNA input:
Template

Total RNA

Small RNA /Circulating RNA

22

D-PLEX SMALL RNA-SEQ KIT MANUAL

Starting amount

UDI library
constructionn cyclesSI library
constructionn cycles

100 ng

10

10

10 ng

12

12

1 ng

14

15

100 pg

18

20

10 ng

10

10

1 ng

13

12

100 pg

15

15

10 pg

18

20

Incubate for PCR amplification according to the following program:
For UDI library construction:
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

n cycles10 minutes

DETAILED PROTOCOL

4.5

Hold at 4°C or freeze until further processing

For SI library construction:
Step

Temperature

Time & Cycles

1. Initial denaturation

98°C

30 seconds

2. Denaturation

98°C

15 seconds

3. Annealing

62°C

30 seconds

4. Extension

72°C

30 seconds

5. Final extension

72°C

n cycles10 minutes

Hold at 4°C or freeze until further processing

CAUTION: If you plan to proceed to the size-selection of the library for short
inserts (<50 nt) (see sections 6), add 2 more cycles to the n cycles determined
earlier.

23

STEP 5DNA library purification
NOTE: It is recommended to purify the amplified library with a spin columnbased method. As an alternative method, a 1.8x SPRI beads clean-up can be
performed.

5.1

Purify the amplified library using the MicroChIP DiaPure kit(Diagenode, C03040001) according to the kit's instructions (follow
the “Protocol for ChIP samples”). Note that the library volume tostart the purification is around 100 µl.

5.2

Perform the elution in the volume displayed below in a 1.5 ml tube
according to the next library processing step:
Table 5: Elution volume after MicroChIP DiaPure purification
Next library processing step

24

DiaPure elution volume

SPRI beads clean up (Section 6A)

50 µl

Agarose gel-cut size-selection (Section 6B)

20 µl

Polyacrylamide gel-cut size-selection (Section 6C)

20 µl

D-PLEX SMALL RNA-SEQ KIT MANUALDNA library clean-up and shaping

DETAILED PROTOCOLSTEP 6
Choose the appropriate procedure to shape the final form of thelibrary before sequencing:• SECTION 6A: SPRI beads clean-up to retain all the libraryfragments.
• SECTION 6B: Agarose gel-cut size selection for the accurate
selection of any length of fragments.
• SECTION 6C: Polyacrylamide gel-cut size selection for theaccurate selection of any length of fragments.

6A – BEADS CLEAN-UP
6A.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.

6A.2

For UDI library construction: Add 50 µl of beads to the 50 µl of
eluted DNA library from section 5 and mix thoroughly by pipetting
up and down or vortexing.
For SI library construction: Add 75 µl of beads to the 50 µl of eluted
DNA library from section 5 and mix thoroughly by pipetting up anddown or vortexing.

25

6A.3

Incubate at least for 5 minutes at room temperature under mild
agitation.

6A.4

Spin down the tube and place it in a magnetic rack until the beads
are collected to the side of the tube and the solution is completely
clear.

6A.5

Carefully remove and discard the supernatant without disturbing
the beads.

6A.6

While keeping the tube on the magnetic rack, perform 2 roundsof bead-wash. Add 200 µl of freshly prepared 80% ethanol for
30 seconds and then remove carefully the supernatant without
disturbing the beads.

6A.7

Spin down the tube, place it back in the magnetic rack and remove
any remaining ethanol.

6A.8

Leave the tube open to let the beads pellet dry for 2 minutes andthen remove it from the magnetic rack.

6A.9

Resuspend the beads in 15 µl of nuclease-free water by slowly
pipetting up and down.

6A.10 Incubate the beads in water during 2-3 minutes at room temperature
under mild agitation.
6A.11 Spin down the tube and place it in the magnetic rack until the beads
collected to the size of the tube and the solution is completely clear.
6A.12 Carefully collect the supernatant without taking up any beads.
Transfer the cleaned-up library in a RNase-free tube and store it
at -20°C until further use.

26

D-PLEX SMALL RNA-SEQ KIT MANUAL

6B.1

Load the 20 µl eluted DNA library from section 5 (mixed withappropriate electrophoresis loading buffer) in a well of a 4%
agarose gel soaked in 0.5X TBE buffer. Use a gel stain of your best
convenience (e.g. 1x SYBR® Gold nucleic acid gel stain or 1x SYBR®
Safe DNA gel stain from Thermo Fisher Scientific) and a ladder ofyour choice (e.g. 25-100bp mixed DNA ladder Bionneer D-1020).

6B.2

Run the electrophoresis until the migration front reaches the endof the gel.

6B.3

View the gel on a UV transilluminator and perform the gel-excisionof the band of interest. For example, the miRNA band is around~195 bp for UDI library construction whereas it is ~180 bp for SI
library construction.

6B.4

Put the gel-slice in a 1.5 ml tube and perform the DNA gel extraction
using the MinElute® Gel extraction kit from Qiagen BUT do notfollow Qiagen's instructions.

6B.5

Add 600 µl of buffer GQ and incubate overnight at 37°C under anagitation of 300 rpm.

6B.6

After gel dissolution, spin down the tube, collect the solution andtransfer it in a MinElute® column.

6B.7

Centrifuge for 20 seconds at 16.000g and discard the flow-through.

6B.8

Add 0.75 ml of buffer PE to the MinElute® column, incubate for 1
minute and centrifuge for 1 minute at 16.000g.

6B.9Discard the flow-through and centrifuge the column for 1 minuteat 16.000g.

DETAILED PROTOCOL

6B – AGAROSE GEL-CUT

6B.10 Place the MinElute® column in a clean tube, add 20 µl of Buffer EB
and incubate for 2 minutes at room temperature.
6B.11 Centrifuge for 1 minute at 10.000g and collect the DNA library in a
RNase-free tube. Store it at -20°C until further use.
27

6C – POLYACRYLAMIDE GEL-CUT
6C.1

Load the 20 µl eluted DNA library from section 5 (mixed withappropriate electrophoresis loading buffer) in a well of a 4-6%
polyacrylamide gel soaked in 0.5xTBE buffer. Use a gel stain ofyour best convenience (e.g. 1x SYBR® Gold nucleic acid gel stain or
1x SYBR® Safe DNA gel stain from Thermo Fisher Scientific) and aladder of your choice (e.g. 25-100bp mixed DNA ladder Bionneer
D-1020).

6C.2

Run the electrophoresis until the migration front reaches the endof the gel.

6C.3

View the gel on a UV transilluminator and perform the gel-excisionof the band of interest. For example, the miRNA band is around~195 bp for UDI library construction whereas it is ~180 bp for SI
library construction.

6C.4

Put the gel-slice in a 1.5 ml tube and perform the DNA gel extraction
using the MinElute® Gel extraction kit from Qiagen BUT do notfollow Qiagen's instructions.

6C.5

Crush the gel slice in the tube with a DNase-free pestle or cut it
into very small pieces and then add 600 µl of buffer GQ. Incubate
overnight at 37°C under an agitation of 300 rpm to let DNA diffuse
out of the gel.

6C.6

Spin down the tube at full speed, collect the solution and transfer
it in a MinElute® column;

6C.7

Centrifuge for 20 secondes at 16.000g and discard the flow-through.

6C.8

Add 0.75 ml of buffer PE to the MinElute® column, incubate for 1
minute and centrifuge for 1 minute at 16.000g.

6C.9

Discard the flow-through and centrifuge the column for 1 minuteat 16.000g.

6C.10 Place the MinElute® column in a clean tube, add 20 µl of Buffer EB
and incubate for 2 minutes at room temperature.
6C.11 Centrifuge for 1 minute at 10.000g and collect the DNA library in a
RNase-free tube. Store it at -20°C until further use.
28

D-PLEX SMALL RNA-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 729

D-Plex Small RNA Construct

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

30

D-PLEX SMALL RNA-SEQ KIT MANUAL

Figure 4: The D-Plex Small RNA-seq SI library construct bears the Truseq (Illumina) Small RNA adapters with single
index (SI) and unique molecular identifiers (UMI). Read = UMI – TSM– Insert – A-tail – 3’ adapter.

31

Sequencing Recommendations
Given the high complexity of D-Plex Small RNA-seq libraries, it is advised
to sequence one library with a minimum of 20 million reads in order tosufficiently cover the library content for downstream analysis.
D-Plex small RNA-seq libraries can be sequenced as regular Illumina
libraries, meaning that they don’t require any specific considerations
regarding phiX spike-in or cluster density optimization. However, werecommend to spike ~1% of phiX in the library pool that is about to besequenced for a quality monitoring purpose.
As the D-Plex Small RNA-seq library should contain short sequences(primarily small non-coding RNAs) and a UMI at the 5’ end of the forward
strand, we highly recommend to use single-end sequencing.
Paired-end sequencing makes the data analysis difficult, unreliable insome cases, and due to the predominantly short fragments it generates
mostly redundant information. If you are interested in longer fragments,
we recommend to increase the read length with single-end sequencing,as this parameter should be suitable to read the complete length of thewhole fragment.

32

D-PLEX SMALL RNA-SEQ KIT MANUALUDI library construction
The D-Plex Small RNA-seq UDI library construct bears the TruSeq(Illumina) HT adapters. In case of a multiplexing scenario, it is therefore
recommended to submit the D-Plex libraries as TruSeq HT libraries toyour sequencing provider.
The complete architecture of the D-Plex Small RNA-seq UDI construct isprovided in Figure 1. The empty library size (adapters + template switch +UMI + A tail) is equal to 172 bp.
SI library construction
The D-Plex small RNA-seq SI library construct bears the TruSeq (Illumina)
Small RNA adapters. In case of a multiplexing scenario, it is therefore
recommended to submit the D-Plex libraries as TruSeq small RNA
libraries to your sequencing provider.
The complete architecture of the D-Plex small RNA-seq SI construct isprovided in Figure 2. The empty library size (adapters + template switch +UMI + A tail) is equal to 155 bp.

33

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 5: D-Plex Small 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) correspondto the UMI sequence. In the fumi-tools package, the copy_umi command
can copy these bases to the correct lines in the fastq file. The tool expects
a fastq 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 length34

D-PLEX SMALL RNA-SEQ KIT MANUAL

of the UMI (to copy from the read to the ID line) as input. Optionally, thecomputational time can be reduced by increasing the number of CPUs to
be used via the threads 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
Trimming
Trimming is mandatory for the reads generated with the D-Plex Small
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) thatwill 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 command
removes the UMI, A-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
command 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.

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

35

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 a very high-complexity library which
often include small RNAs that are not identified yet and therefore would
not map to a transcriptome consisting of only known transcripts. Of
course, in addition to the genome alignment, the mapped reads can be
assigned to known transcripts as well as for expression analysis of the
known genes.
We recommend using the STAR alignment software. To run STAR, youneed to provide the following arguments to the program: the input
trimmed reads data file, the length at which reads were sequenced minus
1, the folder containing the STAR indexed genome (see STAR user guidefor more information on how to prepare the genome), and optionally the
number of 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.
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_

36

D-PLEX SMALL RNA-SEQ KIT MANUAL

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.
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 transcriptomealignment 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
Note that fumi-tools outputs name-sorted bam files, which can be directly
used as input for counting software tools. For another application, you
may want to sort the bam files differently (e.g. sorting by coordinates).

37

Counting
The counting or expression level calculation is the last step of the processing
to generate an expression level matrix. We recommend using MGcount*,a counting tool developed at Diagenode with a suitable annotations filethat include the small RNA transcripts that are object of study. MGcount,built on top of featureCounts, employs a flexible quantification approachto deal with datasets containing multiple RNA biotypes such as D-plex
libraries. Non-coding RNAs varying in length, biogenesis, and function,
may overlap in a genomic region, and are sometimes present in the
genome with a high copy number. Consequently, reads may align equallywell to more than one position in the reference genome or/and align to a
position where more than one annotated transcript is located. MGcountemploys two strategies to quantify these reads. Firstly, it assigns reads
in rounds prioritizing small-RNAs over long-RNAs ensuring the unbiased
read attribution to intergenic and intragenic small RNAs while preventing
host-genes transcription over-estimation. Secondly, MGcount collapses
loci where reads consistently multi-map into communities of loci witha graph-based approach. The resultant communities are groups of
biologically related loci with nearly identical sequence. Subsequently,
quantification of reads is performed at the loci-community level, reducing
multi-alignments ambiguity at individual locus. Ultimately, this strategy
maximizes the transcriptome information analysed and improves theinterrogation of non-coding RNAs. MGcount software repository provides
annotation files for A. thaliana, H. sapiens, M. musculus and C. elegans.
The required input files for MGcount are a .txt file listing the paths to thealignment input files (.bam format) and the annotations file (.gtf format).
The output directory path has to be provided as an input as well.
Example command:
MGcount -T 2 --gtf Homo_sapiens.GRCh38.gtf -outdir outputs --bam_infiles input_bamfilenames.txt
MGcount provides the choice to enable/disable the quantificationof all RNA biotypes included in the annotation file in the form of"communities" as optional parameters for small-RNA (--ml_flag_
small) and long-RNA (--ml_flag_long). Both are enabled by default.
The main output of MGcount is the count_matrix.csv file containing an
38

D-PLEX SMALL RNA-SEQ KIT MANUAL

expression matrix that can be imported to R or any other software fordownstream analyses. A full user guide for MGCount is available here:
https://filedn.com/lTnUWxFTA93JTyX3Hvbdn2h/mgcount/UserGuide.html.

Other standard counting tools such as featureCounts or HTSeq-counts canalso be used alternatively. Given the high complexity of D-Plex libraries,
we recommend having a clear understanding of the scientific question
and the goal of the project before proceeding to the choice of the counting
method as this will strongly impact downstream analyses. Diagenodeprovides bioinformatics support as a service (please, contact us if you
need help with data analysis).
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. Stranded
mode is selected by default in MGcount.
*Hita, A., Brocart, G., Fernandez, A. et al. MGcount: a total RNA-seq quantification tool to address
multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts. BMC
Bioinformatics 23, 39 (2022). https://doi.org/10.1186/s12859-021-04544-3

Links for the tools used in the example pipeline
Tool

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/downloadfeatureCounts

http://subread.sourceforge.net

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

MGcount

https://github.com/hitaandrea/MGcount39

Example of Results
Quality requirements
When the D-Plex Small RNA-seq protocol is followed precisely and theproper conditions for the particular RNA input are used, the following
standards of quality can be expected:
Table 6: Minimum yield expected from a D-Plex library after DiaPure
purification for different inputs and templates.
Template

Total RNA
Small RNA (< 200 nt)

Starting amountDNA quantity after DiaPure
purification (ng)100 pg~50

100 ng

~50

1 ng

~20

D-Plex Small RNA-seq UDI library construct

Figure 6: DNA electropherogram of a D-Plex Small RNA-seq UDI library construction (1.0X clean-up) made from 50 pg
of circulating RNA, isolated from human plasma. 1 ng of the library was loaded on the Bioanalyzer.

40

D-PLEX SMALL RNA-SEQ KIT MANUAL

Figure 7: DNA electrophoregram of a D-Plex Small RNA-seq UDI library construction (1.0X clean-up) made from 100 pg
of commercial human brain RNA (ThermoFisher Scientific, AM7962). 1 ng of the library was loaded on the Bioanalyzer.

D-Plex Small RNA-seq SI library construct

Figure 8: DNA electrophoregram of a D-Plex Small RNA-seq SI library construct (1,5X clean-up) made from 100 pg ofcommercial human brain RNA (Thermo Fisher Scientific, AM7962). 1 ng of the library was loaded on the Bioanalyzer.

41

Related Products
Product

Reference

D-Plex Unique Dual Indexes Module – Set A

C05030021

D-Plex Unique Dual Indexes Module – Set B

C05030022

D-Plex Unique Dual Indexes Module – Set C

C05030023

D-Plex Unique Dual Indexes Module – Set D

C05030024

D-Plex Single Indexes Module - Set A

C05030010

D-Plex Single Indexes Module - Set B

C05030011

DiaMag 0.2 mL tube magnetic rack

B04000001

MicroChIP DiaPure Kit

C03040001

Revision history

42

VersionDate of modification

V2 03_2024

March 2024

- Page 22 - Typo correction in the step 4.4

V2 08_2023

August 2023

- Update of the paragraph "Counting", page 39- Addition of new references and removal of obsolete
references

V2 03_2021

March 2021/

D-PLEX SMALL RNA-SEQ KIT MANUAL

Description of modifications

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