D-Plex 24 DNBSEQ™
BarcodesBarcodes modules for D-Plex Small
RNA DNBSEQ™ Kit
Cat. No. C05030060  (Set A: [4, 4, 8, 8])
C05030061  (Set B: [24])

USER GUIDEV1 12_2020

Summary
Kit Method Overview

4Introduction

5

Kit Materials

6

Multiplexing Advices

8

Related Products 	

10

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

4STEP
5

Library purification

STEP
6

Library clean-up and shaping

STEP
7

Library control
and quantification

D-PLEX 24 DNBSEQ™ BARCODES 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 to100 ng for total RNA.
The core of the technology relies on ligation-free reactions to attach theMGI 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. D-Plex 24 DNBSEQ Barcode sequences – Set A [4, 4, 8, 8]
x-Barcode
Set

D-PlexDNBSEQ
Barcode#PCR Reverse Primer sequence

Barcode

1TGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTAGGTCCGAT

2TGTGAGCCAAGGAGTTGGATTCCGTCCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGACGGAATC

3TGTGAGCCAAGGAGTTGCGGCAGTAAGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTTACTGCCG

4TGTGAGCCAAGGAGTTGTCAATTAGGTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTACCTAATTGA13TGTGAGCCAAGGAGTTGCGGATTGCCGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGGCAATCCG

14TGTGAGCCAAGGAGTTGGAATCCTGATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTATCAGGATTC15TGTGAGCCAAGGAGTTGTCTGGAATGATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCATTCCAGA16TGTGAGCCAAGGAGTTGATCCAGCATCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGATGCTGGAT57TGTGAGCCAAGGAGTTGTCCGTTGAATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTATTCAACGGA58TGTGAGCCAAGGAGTTGCAGTACAGTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAACTGTACTG

59TGTGAGCCAAGGAGTTGATTGAGGTACTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTACCTCAAT60

TGTGAGCCAAGGAGTTGATTAGAAGTCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT

GACTTCTAAT61

TGTGAGCCAAGGAGTTGCAACGCTTCATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGAAGCGTTG

62TGTGAGCCAAGGAGTTGGGATCGCACGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGTGCGATCC63

TGTGAGCCAAGGAGTTGTGCCTTCCGATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCGGAAGGCA64TGTGAGCCAAGGAGTTGGCGACATCGGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT

CCGATGTCGC

65TGTGAGCCAAGGAGTTGCATTCTAAGTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTACTTAGAATG66

TGTGAGCCAAGGAGTTGCAGGCTTGGATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCCAAGCCTG

67

TGTGAGCCAAGGAGTTGATCATCGTCTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAGACGATGAT68

TGTGAGCCAAGGAGTTGGTCTTGTGAGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTCACAAGAC

69

TGTGAGCCAAGGAGTTGAGTAGGAACGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGTTCCTACT70TGTGAGCCAAGGAGTTGTCACAACCACTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT

GTGGTTGTGA

71TGTGAGCCAAGGAGTTGGCAGGCCTTCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGAAGGCCTGC72TGTGAGCCAAGGAGTTGTGGCAAGCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT

TAGCTTGCCA

44886

D-PLEX 24 DNBSEQ™ BARCODES MANUALTable 2. D-Plex 24 DNBSEQ Barcode sequences – Set B [24]x-BarcodeSet

D-PlexDNBSEQBarcode#PCR Reverse Primer sequence

Barcode25TGTGAGCCAAGGAGTTGTTGTCCTCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTAGAGGACAA

26TGTGAGCCAAGGAGTTGATTCGCTAGGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCTAGCGAAT

28TGTGAGCCAAGGAGTTGACAGCTCAGCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTGAGCTGT29TGTGAGCCAAGGAGTTGTATCTAGGTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT

AACCTAGATA

30TGTGAGCCAAGGAGTTGGAGATGGCAATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTGCCATCTC32TGTGAGCCAAGGAGTTGGCCGATAGCGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGCTATCGGC

33TGTGAGCCAAGGAGTTGCCATCGTTGCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCAACGATGG

34TGTGAGCCAAGGAGTTGTGAACGATTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTAATCGTTCA

35TGTGAGCCAAGGAGTTGTAGAGCGAACTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGTTCGCTCTA

36TGTGAGCCAAGGAGTTGATGTGTGAGATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTCTCACACAT37

TGTGAGCCAAGGAGTTGATCCTAACAGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCTGTTAGGAT38TGTGAGCCAAGGAGTTGCGCGTCTGCGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCGCAGACGCG

39TGTGAGCCAAGGAGTTGGATGATCCTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAAGGATCATC49

TGTGAGCCAAGGAGTTGCTGCGTACATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTATGTACGCAG

50TGTGAGCCAAGGAGTTGATCTCATTAATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTTAATGAGAT

51TGTGAGCCAAGGAGTTGAAGTGGCGCATTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGCGCCACTT52

TGTGAGCCAAGGAGTTGGGCCTTAATGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCATTAAGGCC53TGTGAGCCAAGGAGTTGTCTGAGGCGGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT

CCGCCTCAGA

55

TGTGAGCCAAGGAGTTGGATAACCGGCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCCGGTTATC56TGTGAGCCAAGGAGTTGTCAATATTCCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGGAATATTGA

114

TGTGAGCCAAGGAGTTGCTGACTCTGGTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTCCAGAGTCAG

115

TGTGAGCCAAGGAGTTGACTGCCTGTTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTAACAGGCAGT116TGTGAGCCAAGGAGTTGGTCATGGAGCTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTGCTCCATGAC

117TGTGAGCCAAGGAGTTGGGATAGACATTTGTCTTCCTAAGACCGCTTGGCCTCCGACTTATGTCTATCC247Table 3. Kit content
Quantity

Storagetemperature

D-Plex Forward Primer DNBSEQ (FP) (x1)

240 μl

-20°C/4°F

D-Plex Reverse Primer DNBSEQ (RP) (x24)

30 μl each

-20°C/4°F

Reagent

Multiplexing Advices
The listed PCR Reverse Primers in Tables 1 and 2 are bearing the MGIEasy
barcodes that can be used for library multiplexing up to 48. In case ofa multiplexing scenario, it is then recommended to follow MGI’s librarypooling guidelines that are summarized in Table 4.
Table 4. Multiplexing recommendations for the D-Plex Small RNA DNBSEQ kit
Instruction

Level ofmultiplexing

1(Mixtures need to be prepared with equal volumes of each barcode
to use them in a final volume of 10 µl for the PCR reaction)
1. Mix barcoded Reverse Primers of a 4-Barcode Set and give it to the sample.
2. Mix barcoded Reverse Primers of a 8-Barcode Set and give it to the sample.
1. P repare 2 different mixtures with 2 different barcoded Reverse Primers of a 4-Barcode Set and2give one mixture to one sample.
2. Prepare 2 different mixtures with 4 different barcoded Reverse Primers of a 8-Barcode Set andgive one mixture to one sample.
For sample 1 & 2, use the method for 2-sample multiplexing above.

3

For sample 3, use the method for 1-sample multiplexing above.
Note that you should use different Barcode Sets for samples 1, 2 and 3.
1. Use a different barcoded Reverse Primers of a 4-Barcode Set for each sample.

42. Prepare 4 different mixtures with 2 different barcoded Reverse Primers of a 8-Barcode Set andgive one mixture to one sample.
For samples 1-4, use the method for 4-sample multiplexing above.

5

For sample 5, use the method for 1-sample multiplexing above.
Note that you should use different Barcode Sets for samples 1-4 and 5.
For samples 1-4, use the method for 4-sample multiplexing above.

6

For samples 5-6, use the method for 2-sample multiplexing above.
Note that you should use different Barcode Sets for sample 1-4 and 5-6.

8D-PLEX 24 DNBSEQ™ BARCODES MANUALInstruction

Level ofmultiplexing(Mixtures need to be prepared with equal volumes of each barcode
to use them in a final volume of 10 µl for the PCR reaction)

1. For samples 1-4, use the method for 4-sample multiplexing above.
(Use 1st Barcode Set).
2. For samples 5-6, use the method for 2-sample multiplexing above.

7(Use 2nd Barcode Set).
3. For sample 7, use the method for 1-sample multiplexing above.
(Use 3rd Barcode set)
Note that you should use different Barcode Sets for samples 1-4, 5-6 and 7.

88n+X(1≤n≤2;X=1-8;total 17-24)

Use a different barcoded Reverse Primers of a 8-Barcode Set for each sample.

1. For samples 1-8, use the method for 8-sample multiplexing above.
2. For samples 9-8n, use the method for 8-sample multiplexing above.
3. For samples 8n+1 - 8n+X, according to the value of X, use the methods above for X-sample
multiplexing accordingly. Remember to use different Barcode Sets.
Note that you should use different Barcode sets for steps 1), 2) and 3).

1. For samples 1-24 use a different barcoded Reverse Primer of the 24-Barcode Set for eachsample.

8n+X (3≤n≤5;X=1-8;
total 25-48)

2. For samples 25-8n, separate the samples into groups of 8, and use the method for 8-sample
multiplexing above. Remember to use different Barcode Sets.
3. For samples 8n+1 - 8n+X, according to the value of X, use the methods above for X-sample
multiplexing accordingly. Remember to use different Barcode Sets.
Note that you should use different Barcode Sets for steps 1), 2) and 3).

9Related products
Indexes Module Reference

10Reference

D-Plex Small RNA DNBSEQ™ Kit

C05030059

DiaMag 0.2 mL tube magnetic rack

B04000001

DiaMag 1.5 mL tube magnetic rack

B04000003

MicroChIP DiaPure columns

C03040001

D-PLEX 24 DNBSEQ™ BARCODES MANUALFOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
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