D-Plex Small RNA
DNBSEQ™ Kit
Small RNA library preparation kit
for MGI® sequencers
Cat. No. C05030051  (Core module – 24 rxns)

USER GUIDE
V2

04_2021

Summary
Kit Method Overview

4

Introduction

5

Kit Materials

6

Required Materials Not Provided

7

Remarks Before Starting

9

Short Protocol (for experienced users)

13

Detailed Protocol

17

D-Plex Small RNA DNBSEQ Construct

29

Sequencing Recommendations

30

Data Analysis Recommendations

32

Example of Results

36

Related Products 	

38

Please read this manual carefully
before starting your experiment
Barcodes are not included in this kit and areavailable separately. Please check “Required
Materials Not Provided” section.

Kit Method Overview
Time Pause

RNA

STEP
1

RNA

STEP
2

AAAAA...

RNA
5’ adaptor

STEP
3

RNA sample
preparation

rXrXrXrX

20 minRNA dephosphorylationand tailing

1h30

Reverse transcription>2h

PCR amplification

60 minAAAAAAA...V TTTTTTTT 3’ adaptor...XXXXAAAAAAA
TTTTTTTT

XXXX
XXXX

AAAAAAA
TTTTTTTTPCR forward
PCR reverse

STEP
4

AAAAAAA
TTTTTTTT

XXXX
XXXX

DNA

4

STEP
5

Library purification

STEP
6

Library clean-up and shaping

STEP
7

Library control
and quantification

D-PLEX SMALL RNA DNBSEQ KIT MANUALIntroduction
The Diagenode D-Plex Small RNA DNBSEQ™ Library Preparation Kit is atool designed for the study of the small non-coding transcriptome. The
present kit incorporates the unique D-Plex technology to generate doublestranded DNA libraries from RNA samples ready to be used for the DNA
single-strand circularization step required for DNBSEQ sequencing on
MGI sequencers.
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• Ease of use in a one day, one tube protocol• High library complexity
The library preparation protocol works on either total intact RNA (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 liquidbiopsy-type of sample (blood serum and plasma). The input requirementsof the method are flexible and allow the user to perform the method within
a wide range of RNA quantities going from 10 pg of circulating RNA up to
100 ng for total RNA.
The core of the technology relies on ligation-free reactions to attach the
MGI adaptors to both ends of the library construct. Therefore, the results
generated with the D-Plex Small RNA DNBSEQ kit will vastly differ fromthose produced with a ligase-based approach. For instance, the results
generated with a D-Plex kit will encompass a vast spectrum of small noncoding RNAs (miRNAs, snoRNAs, snRNAs, piRNAs) whereas a ligasebased 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

Kit Materials
Table 1. Components of D-Plex Small RNA DNBSEQ Kit
Description

6

Cap color

Qty (24 rxns)

Storage (°C/°F)

Dephosphorylation Buffer (DB)Yellow

48 µl-20°C/-4°F

Dephosphorylation Reagent (DR)Yellow

12 µl-20°C/-4°F

Tailing Reagent (TR)

Red

12 µl

-20°C/-4°F

Small Tailing Buffer (STB)

Red

24 µl

-20°C/-4°F

RT Primer H DNBSEQ (RTPH)

Purple24 µl-20°C/-4°F

RT Primer M DNBSEQ (RTPM)

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

Template Switching Oligo DNBSEQ (TSO)

Purple48 µl-20°C/-4°F

PCR Master Mix (PCRMM)

Green

1200 µl-20°C/-4°F

Positive Control miRNA (CTL+)

Black

12 µl
(1 ng/ µl)-20°C/-4°F   For long term
storage:-80°C/-112°F

Nuclease-free Water

Clear

2000

Room Temperature

D-PLEX SMALL RNA DNBSEQ KIT MANUALRequired Materials Not Provided
BarcodesSpecific D-Plex DNBSEQ Barcodes were designed and validated to fit thistechnology and are not included in this core module, providing you withtotal flexibility. They can be bought separately according to your needs.
Please choose the format that suits you best among the compatiblereferences to:Table 2. DNBSEQ barcode sets and references
Barcodes Modules

Reference

D-Plex 24 DNBSEQ Barcodes - Set A

C05030060

D-Plex 24 DNBSEQ Barcodes - Set B

C05030061

Multiplexing
The validated PCR reverse primers bear the MGIEasy DNA Barcodes thatcan be used for library multiplexing up to 48. In case of a multiplexing
scenario, it is recommended to follow MGI’s library pooling guidelinesthat are explained in the D-Plex DNBSEQ Barcodes manual.
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 (ThermoFisher
Scientific, 10328011)• 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)• DiaMag 1.5 ml tube magnetic rack (Diagenode, B04000003)• Thermal cycler
7

Post-PCR libraries purification• MicroChIP DiaPure columns (Diagenode, C03040001)• Absolute ethanol (VWR, 20821.310)• DNase-free, RNase-free ultrapure water
Post-PCR libraries shaping• Agencourt® AMPure® XP Beads (Beckman Coulter, A63881)• Absolute ethanol (VWR, 20821.310)• DNase-free, RNase-free ultrapure water
Precise library size-selection with agarose/polyacrylamide gel-cut
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 Agilent High Sensitivity
DNA Kit (Agilent, 5067-4626)• Qubit® Fluorometer (Thermo Fisher Scientific) and Qubit® dsDNA
HS Assay Kit (Thermo Fisher Scientific, Q32851)
Sequencing• MGIEasy Circularization Kit (MGI, 1000005259)• DNBSEQ-G400RS High-throughput Sequencing Set (Small RNAFCL SE50) (MGI, 1000016998)• CoolMPS High-throughput Sequencing Set (DNBSEQ-G400RS
Small RNA FCL SE50) (MGI, 1000019478)

8

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

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• When mixing reagents, do not vortex but pipet up and down
• When using the protocol for the first time for a definite typeof sample, we strongly recommend using the positive miRNA
control included in the kit
Important notes
The D-Plex Small RNA DNBSEQ protocol has been developed for inputs
ranging from 100 ng to 100 pg of total RNA (RIN≥8) and inputs ranging
from 10 ng to 10 pg of small RNA (<200 nt) either isolated from cells or
biological fluids, extracellular vesicles, or others.
A starting volume of 13 μl is used in the library preparation process. RNA
has to be dissolved in nuclease-free water in this volume before starting
the protocol.
It is advised, when performing the protocol for the first time with a particulartemplate, to include a reaction with the positive control provided in the
kit and a reaction without template as negative control. This negative
control is useful to check for the absence of contaminants in the working
environment.

9

The D-Plex Small RNA DNBSEQ library preparation incorporates
the different classes of small non-coding RNAs in the final library forsequencing. If coding RNA or long non-coding RNA degradation products
are of particular interest to the researcher, it is possible to perform an
optional step (described in the detailed protocol, step 2.1) that will help
enrich for those products in the library.
The following protocol describes the library preparation of one single
sample. If the interest is to prepare more than one sample in parallel,
please scale up accordingly.
CAUTION: The incubations during the D-Plex Small RNA DNBSEQ library
preparation take place in a thermal cycler with a heated lid at 105°C.

Starting material
The input used for the D-Plex library preparation needs to be free ofcontaminants and the RNA must be solubilized in nuclease-free water.
Table 3. Input recommendations

10

Template

Starting amount

RIN

Total RNA

100 pg - 100 ng≥8

Small RNA (<200nt)

10 pg - 10 ng

N/A

Circulating RNA

10 pg - 10 ng

N/A

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

Template
Both total RNA and small RNA (<200nt) can be used for input in the D-Plex
Small RNA DNBSEQ library preparation.
Small RNAs can be isolated either from biological fluids (e.g. blood plasma
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 (<200nt). Please be aware that not all the products on the market
are able to efficiently isolate RNAs under 200nt.
For total RNA samples, we strongly recommend to first perform aquality check of the RNA using the Bioanalyzer® 2100 instrument withthe associated Agilent RNA 6000 Pico Kit (Agilent, 5067-1531). Please beaware that sample degradation may result in the underrepresentation of
the small non-coding RNAs in the final library.
The D-Plex method offers a protocol in which the RNA sample is treatedwith a dephosphorylation reagent (DR) that will dephosphorylate the 3’endof an RNA molecule (if it is phosphorylated) and monophosphorylate the
5’end (if it is hydroxylated). This protocol might constitute a special interestfor researchers studying the degradation products of coding RNAs andlong non-coding RNAs.

11

Positive control: cel-miR-39-3p
A positive control is supplied in the kit for 12 reactions at a concentration of
1 ng/µl. This synthetic miRNA is meant to be used in a library preparation
set to monitor the library preparation efficiency or as a spike-in control
in a given RNA sample to account for eventual matrix effect. Typically,
1 ng of synthetic miR39 prepared with the D-Plex protocol and amplified
for 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 22nt long.
5’-UCACCGGGUGUAAAUCAGCUUG-3’

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

12

D-PLEX SMALL RNA DNBSEQ KIT MANUALSHORT
PROTOCOL
FOR EXPERIENCED USERS

Short Protocol for experienced users
Notice before starting
D-Plex Small RNA DNBSEQ library preparation is incorporating 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 ofparticular interest to the researcher, it is possible to perform an optional (step 3) that will help enrich for those
products in the library.

1.

Prepare the RNA sample in nuclease-free water to get a total volumeof 13 µl.

2.

Add 2 µl of Dephosphorylation Buffer (DB) and incubate for 10
minutes at 70°C. Then immediately put on ice for 2 minutes.

3.

OPTIONAL [To proceed to RNA 3’-end dephosphorylation, add 0.5 µl
of Dephosphorylation Reagent (DR) to the reaction tube. Incubate
for 15 minutes at 37°C. Then put on ice].

4.

Prepare the Tailing Master Mix (TMM) by mixing 0.5 µl of Tailing
Reagent (TR) and 1 µl of Small Tailing Buffer (STB).

5.

Add 1.5 µl of TMM and incubate for 40 minutes at 37°C + 20 minutes
at 65°C. Then put on ice to cool down.

6.

Add 1 µl of Reverse Transcription Primer DNBSEQ (RTP):• For 100 ng-10 ng total RNA or 10 ng-1 ng small RNA fraction, use
RT Primer H DNBSEQ (RTPH).
• For 10 ng-100 pg total RNA or 1 ng-10 pg small RNA fraction, use
RT Primer M DNBSEQ (RTPM).

14

7.

Incubate for 10 minutes at 70°C. Then slowly decrease to 25°C for 2
minutes by ramping down at 0.5°C/sec.

8.

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

9.

Prepare the Reverse Transcription Master Mix (RTMM) by mixing
5µl of Reverse Transcription Buffer (RTB) and 1µl of Reverse
Transcription Reagent (RTR) in a nuclease-free PCR tube.

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

Add 6µl of Reverse Transcription Master Mix (RTMM) and incubate
for 15 minutes at 25°C.

11.

Add 2 µl of Template Switch Oligo DNBSEQ (TSO).

12.

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

13.

Add 10 µl of D-Plex Forward Primer DNBSEQ (FP), 10 µl of D-Plexbarcoded Reverse Prime DNBSEQ (RP), and 50 µl of PCR MasterMix (PCRMM).

14.

Amplify the library according to the following program:Temperature

Time & Cycles

98°C

30 seconds

98°C

15 seconds

62°C

30 seconds

72°C

30 seconds

72°C

SHORT PROTOCOL

10.

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 RNA or 10 ng small RNA/circulating RNA- up to 20 cycles: for 100 pg total RNA or 10 pg small RNA/circulating RNA

15.

Purify the PCR product using the MicroChIP DiaPure Kit (Diagenode,
C03040001), according to the kit instructions. To perform further
clean-up or size-selection of the library, refer to sections 6-A-B-Cof 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.

15

16

D-PLEX SMALL RNA DNBSEQ KIT MANUALDE TAIL ED
PROTOCOL
STEP 1 - RNA sample preparation

18

STEP 2 - RNA dephosphorylation and tailing

19

STEP 3 - Reverse transcription

20

STEP 4 - PCR amplification

22

STEP 5 - Library purification

23

STEP 6 - Library clean-up and shaping

24

STEP 7 - Library quality control and quantification

28

STEP 1
RNA sample preparation -18

20 minutes

1.1

Dilute the RNA sample in nuclease-free water to get a total volumeof 13 µl.

1.2

To the 13 µl RNA solution, add 2 µl of Dephosphorylation Buffer (DB).
Mix by pipetting up and down thoroughly but gently until solution ishomogenous.

1.3

Incubate for 10 minutes at 70°C and then immediately put the tube on
ice for 2 minutes. Spin down the reaction tube to collect the sample
at the bottom.

D-PLEX SMALL RNA DNBSEQ KIT MANUALRNA dephosphorylation and tailing
1 hour 30 minutes

2.1

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

2.2

Prepare the Tailing Master Mix (TMM) by mixing 0.5 µl of Tailing
Reagent (TR) and 1 µl of Small Tailing Buffer (STB) in a tube.

2.3

Add 1.5 µl of TMM to the reaction tube and mix by pipetting up anddown until solution is homogeneous.

2.4

Incubate the sample for 40 minutes at 37°C + 20 minutes at 65°C andthen put the tube on ice to let it cool down. Spin down the reaction
tube to collect the sample at the bottom.

DETAILED PROTOCOLSTEP 219

STEP 3
Reverse transcription 3.1> 2 hours

Add 1 µl of Reverse Transcription Primer DNBSEQ (RTP) to thereaction tube. Choose the right RTP according to your RNA input:
Table 4: RT primer concentration according to RNA input
Template
Total RNA
Small RNA (<200nt)
Circulating RNA

20

Small RNA fraction / biofluid-isolated RNA
100 pg –  10 ng

RTPH

10 ng - 100 ng

RTPM

10 pg - 1 ng

RTPH

1 ng - 10 ng

RTPM

10 pg - 1 ng

RTPH

1 ng - 10 ng

RTPM

3.2

Mix by pipetting up and down until solution is homogeneous. Incubate
for 10 minutes at 70°C and then slowly decrease to 25°C by ramping
down at 0.5°C/sec (maintain to 25°C for 2 minutes). Spin down thereaction tube to collect the sample at the bottom and then put the
tube on ice until further processing.

3.3

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

3.4

Prepare the Reverse Transcription Master Mix (RTMM) by mixing
5µl of Reverse Transcription Buffer (RTB) and 1µl of Reverse
Transcription Reagent (RTR) in a nuclease-free PCR tube.

3.5

Add 6µl of Reverse Transcription Master Mix (RTMM) to thereaction tube and mix by pipetting up and down until the solution ishomogenous.

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

Incubate for 15 minutes at 25°C and then put the tube on ice.

3.7

Add 2 µl of Template Switch Oligo DNBSEQ (TSO) to the reaction tube
and mix by pipetting up and down until the solution is homogeneous.

3.8

Incubate the sample for 120 minutes at 42°C + 10 minutes at 70°C
and then put the tube 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 firsttwo steps are completed.

3.9

DETAILED PROTOCOL

3.6

Spin down the reaction tube to collect the sample at the bottom.

21

STEP 4
PCR amplification -1 hour

4.1

Add 10 µl of D-Plex Forward Primer DNBSEQ (FP) and 10 µl of
D-Plex barcoded Reverse Primer DNBSEQ (RP) to the reaction tube.
Add 50 µl of PCR Master Mix (PCRMM) and mix directly by pipetting
up and down until solution is homogenous.

4.2

Determine the number of PCR cycles (n) you have to perform,
according to the RNA input:
Table 5: PCR amplification program for D-Plex Small RNA DNBSEQ
library preparation
Input RNA

Total RNA

Small RNA fraction / Fluid-isolated RNA

4.3

Amount RNAn

100 pg

20

1 ng

15

10 ng

12

100 ng

10

10 pg

20

100 pg

15

1 ng

12

10 ng

10

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

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-B-C), add 2 more cycles to the n cycles displayed
above.
22

D-PLEX SMALL RNA DNBSEQ KIT MANUAL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 (see section 6A).

5.1

Purify the amplified library using the MicroChIP DiaPure kit(Diagenode, C03040001) according to the kit instructions. Note thatthe library volume to start 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:

DETAILED PROTOCOLSTEP 5

Table 6: Elution volume after MicroChIP DiaPure purification
Next library processing step

DiaPure elution
volumeNo further clean-up or size selection

20 µL

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

23

STEP 6
Library clean-up and shaping
NOTE: To purify the libraries, the MicroChip DiaPure purification method should
be sufficient and no additional clean-up is required. However, if necessary or
preferred, an additional 1.8x SPRI beads clean-up can be performed to remove
residual oligos (Section 6.A).

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

24

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

Add 90 µl of beads to the 50 µl of eluted DNA from Step 5 and mix
thoroughly by pipetting up and down or vortexing.

6A.3

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

6A.4

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

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

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 2-3 minutes andthen remove it from the magnetic rack.

6A.9

Resuspend the beads in 20 µl of nuclease-free water by slowly
pipetting up and down.

DETAILED PROTOCOL

6A.5

6A.10 Incubate the beads in water during 2 minutes at room temperature
under mild agitation.
6A.11 Spin the tube down 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 nuclease-free tube and store it
at -20°C for further use.

6B – AGAROSE GEL-CUT SIZE-SELECTION
6B.1

Load your 20 µl eluted DNA library from step 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~120 bp.
25

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 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 the tube down, collect the solution totransfer 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.

6B.10 Place the MinElute® column in a clean 1.5ml tube, add 20 µl of
Buffer EB and incubate 2 minutes at room temperature.
6B.11 Centrifuge for 1 minute at 10.000g and collect the eluted DNA
library. Store at -20°C for further use.

6C – POLYACRYLAMIDE GEL-CUT SIZE-SELECTION

26

6C.1

Load your 20 µl eluted DNA library from step 5 (mixed withappropriate electrophoresis loading buffer) in a well of a 4-6%
polyacrylamide gel soaked in 2xTBE 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~120 bp.

D-PLEX SMALL RNA DNBSEQ KIT MANUAL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 and incubate
overnight at 37°C under a 300 rpm agitation to let DNA diffuse outof the gel.

6C.6

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

6C.7

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

6C.8

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

6C.9

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

DETAILED PROTOCOL

6C.4

6C.10 Place the MinElute® column in a clean 1.5ml tube, add 20 µl of
Buffer EB and incubate 2 minutes at room temperature.
6C.11 Centrifuge 1 minute at 10.000g and collect the eluted DNA library.
Store at -20°C for further use.

27

STEP 7
Library quality control andquantification

28

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 HighSensitivity assay kit according to the manufacturer’s instructions.

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

= PolyA and PolyT tail
= DNBSEQ Barcode
B= MGI 3’ adaptor
3’

Poly A/T= MGI 5’ adaptor

INSERTTSM
5’

5’INSERTTSM
5’

TSM= Templates-Switch Motif (3nt)

3’
PolyT

PolyA

3’

B

D-Plex Small RNA DNBSEQ Construct

Figure 2: The D-Plex Small RNA DNBSEQ construct bears the MGIEasy DNA Barcodes allowing library multiplexing up to 48.

29Sequencing Recommendations
The D-Plex Small RNA DNBSEQ Kit allows the production of dsDNA
libraries, which are designed for DNBSEQ sequencing on MGI sequencers.
Therefore, a D-Plex Small RNA DNBSEQ library pool needs to undergo acircularization reaction prior to the subsequent DNB-production, flowcell loading and sequencing procedure. For this circularization step, we
refer to the MGIEasy Circularization Kit (MGI, 1000005259).
To obtain high quality data, we recommend the MGI DNBSEQ-G400
sequencing system with the usage of the new CoolMPS technology(CoolMPS High-throughput Sequencing Set (DNBSEQ-G400RS Small
RNA FCL SE50) (MGI, 1000019478))
The D-Plex Small RNA DNBSEQ library construct bears the MGIEasy
DNA Barcodes that can be used for library multiplexing up to 48. In caseof a multiplexing scenario, it is then recommended to follow MGI’s librarypooling guidelines that are explained in the D-Plex Small RNA DNBSEQ
Barcodes manual.
The complete architecture of the D-Plex Small RNA DNBSEQ construct isprovided in Figure 2. The empty library size (adapters + template switch +A tail) is equal to 108 bp.
IMPORTANT NOTICES:- Given the high complexity of D-Plex Small RNA DNBSEQ libraries, it isadvised to sequence one library with a minimum of 20M (millions) SE(single-end) reads in order to sufficiently cover the library content for
downstream analysis.
- Since the library sequence starts with the template switch motif, werecommend to initiate the sequencing with 3 dark cycles.

30

D-PLEX SMALL RNA DNBSEQ KIT MANUALTable 7: Processing and sequencing recommendations
ssDNA circularization

MGIEasy Circularization Kit (MGI, 1000005259)

Sequencing system

MGI DNBSEQ-G400

Sequencing set

DNBSEQ-G400RS High-throughput Sequencing Set(Small RNA FCL SE50) (MGI, 1000016998)

Sequencing chemistry

CoolMPS High-throughput Sequencing Set (DNBSEQG400RS Small RNA FCL SE50) (MGI, 1000019478)

Sequencing parameters

SE50, dark cycles 1-3

31Data Analysis Recommendations
The following is a guideline to allow you to start analyzing your D-Plexsequenced data. Depending on the aim of your analysis you might need totry different parameters and tools.
The D-Plex libraries contain special sequences that need particulartreatment in order to get the best results out of your datasets. These
special sequences are the poly-A tail and the template switch motif. This
guide will take you through the basic processes of trimming, alignmentand counting, using software tools and settings that we validated. Thoughnaturally other tools and methods can also be used, please pay attention
to finding the optimal settings for your experiments, e.g. do not run apaired-end pipeline for single reads.
Main pipeline
TRIMMING

ALIGNMENT

COUNTING

NOTE: The links for the tools used in the example pipeline are at the end of thissection.

Trimming
Trimming is mandatory for the reads generated with the D-Plex Small RNA
DNBSEQ Kit, because the read length is often longer than the targeted
small RNAs, so in many cases the poly-A tail and the 3’ adapter are alsosequenced. In addition, the 5’ end contains the template switch motif thatis originally not part of the RNA. The aim of trimming is to remove these
artificial sequences that will likely hamper downstream analyses.
In our example below, we use cutadapt to demonstrate how to properly
do the trimming for D-Plex reads and get a read set as clean as possible.
To minimize the effect of ambiguously read bases, we trim the Ns
and allow IUPAC codes in the reads. We then remove   the 3’ adapter(AAGTCGGAGGCCAAGCGGTCT) and the poly-A tail from the 3’ end of the read.
Finally, we remove the 5’ adapter (GAACGACATGGCTACGATCCGACTTSSS)
from the 5’ end of the read. We found that this combination of parameters
32

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

prevents excessive over-trimming and read loss, while reliably and fullycleans up the read set coming from an average MGI sequencing run. The
last step includes discarding reads below 15 nucleotides, as they might bedifficult to map uniquely. The example command with cutadapt is below.
cutadapt --trim-n -a AAGTCGGAGGCCAAGCGGTCT raw_reads.fastq.gz| cutadapt --match-read-wildcards -a AAAAA - | cutadapt -O 8--match-read-wildcards -g GAACGACATGGCTACGATCCGACTTSSS -m 15-o trimmed_reads.fastq.gz –Alignment
Aligning the trimmed reads needs special treatment as mapping small
reads requires the use of specialized aligners that are suitable formapping small RNA DNBSEQ reads. We strongly recommend aligningto the genome instead of the transcriptome. Indeed, D-Plex tends togenerate very high-complexity libraries 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 genomealignment, the mapped reads can be assigned to known transcripts aswell for expression analysis of the known genes.
In our example command below, we use the software STAR which does
exactly that: it aligns the reads to the reference genome, then assigns
them to known transcripts, using a GTF annotation file. For this example,
we assume that the reads were 50 bases long and STAR was run in
multithreading mode on 10 cores. NOTE: for mapping any type of unspliced
RNA, STAR recommends to index the genome without the GTF file, sinceSTAR only uses splice junctions information for mapping.
STAR --runThreadN 10 --genomeDir /genomes/hg19/--readFilesIn trimmed_reads.fastq.gz --sjdbGTFfile genomes/
hg19/hg19.gtf --alignIntronMax 1 --readFilesCommand zcat--outFileNamePrefix ./MySample --outSAMtype BAM Unsorted--outFilterMultimapScoreRange 0 --outFilterMultimapNmax 50--outFilterMismatchNoverLmax 0.05 --outFilterMatchNmin 15--outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread
0 --sjdbOverhang 49 --quantMode GeneCounts TranscriptomeSAM--quantTranscriptomeBAMcompression -1 --outSAMunmapped Within33

CountingIn our command example below, we use the featureCounts program fromthe Subread package for counting. The other options specify that we wantto keep multimapping reads, since some small RNA biotypes map tomultiple loci. Each read will carry a fractioned count (option –fraction).
The -O option will allow a read to be assigned to more than one matchedmeta-feature (this is optional). In the end we will obtain the expression
levels of annotated features as unnormalized values (counts).
featureCounts -a genomes/hg19/hg19.gtf -o MySample_
featureCounts.txt -T 10 -M -O --fraction MySample_sorted.bam

34

D-PLEX SMALL RNA DNBSEQ KIT MANUALLinks for the tools used in the example pipeline
Tool

Link

Websitehttps://www.diagenode.com/en/

Cutadapt

https://github.com/marcelm/cutadapt

STAR

https://github.com/alexdobin/STAR

Samtools

http://www.htslib.org/downloadfeatureCounts

http://subread.sourceforge.net/

35

Example of Results
Quality requirements
When the D-Plex Small RNA DNBSEQ protocol is followed accordinglywith the appropriate conditions for the  RNA input, the following standardsof quality can be expected :Table 8: Minimum yield expected from a D-Plex Small RNA DNBSEQ
library after DiaPure purification for different inputs and templates.
Input

DNA quantity after DiaPure
purification (ng)Total RNA (from K562 cells)

100 ng~100 ng

Total RNA (from K562 cells)

100 pg~100 ng

Small RNA (<200nt)

1 ng~50 ngTemplate

Electrophoregrams

Figure 3: DNA electropherogram of a D-Plex Small RNA DNBSEQ library (DiaPure purification) made from 25 ng of K562 total RNA. 1 ng of thelibrary was loaded on the Bioanalyzer.

36

D-PLEX SMALL RNA DNBSEQ KIT MANUAL

Figure 4: DNA electropherogram of a D-Plex Small RNA DNBSEQ library (DiaPure purification) made from 500 pg of K562 total RNA. 1 ng of thelibrary was loaded on the Bioanalyzer.

Figure 5: DNA electropherogram of a D-Plex-MGI small RNA library (DiaPure purification) made from 50 pg of circulating RNA, isolated fromhuman plasma. 1 ng of the library was loaded on the Bioanalyzer.

37

Related Products
Indexes Module Reference

38

Reference

D-Plex Small RNA DNBSEQ Barcodes - Set A

C05030060

D-Plex Small RNA DNBSEQ Barcodes - Set B

C05030061

DiaMag 0.2 mL tube magnetic rack

B04000001

DiaMag 1.5 mL tube magnetic rack

B04000003

MicroChIP DiaPure columns

C03040001

D-PLEX SMALL RNA DNBSEQ KIT MANUALFOR RESEARCH USE ONLY.
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TRADEMARKSThe trademarks mentioned herein are the property of Diagenode or their respective owners.
Bioanalyzer is a trademark of Agilent Technologies, Inc. Agencourt and AMPure are registeredtrademarks of Beckman Coulter, Inc. DNBSEQ is a trademark of Complete Genomics, Inc. M MGI
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39

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