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Instruction manual for GenNext RamDA-seq Single Cell Kit 202011

GenNext™ RamDA-seq™ Single Cell Kit
RMD-101T 96 reactions
RMD-101TT 24 reactions

Store at −20°C

Contents[1]

Introduction[2]

Components[3]

Protocol
1. Cell lysis or RNA Dilution
2. Denaturation
3. Digestion of genomic DNA
4. cDNA synthesis and amplification (RT-RamDA™)
5. Second-strand synthesis
6. Library preparation
7. Library QC
8. Sequencing QC[4]Application data

[5]

Troubleshooting[6]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory safety precautions while using this kit.
* Illumina™ and Nextera™ are trademarks of Illumina, Inc* Agencourt and AMPure are trademarks of Beckman Coulter, Inc* MultiNA™ is a trademark of the Shimadzu Corporation.
* RamDA-seq™ is a trademark of RIKEN, Institute of Physical and Chemical Research.
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www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
The RamDA-seq™ Single Cell Kit is a kit for preparing cDNA for subsequent NGS
analysis from single cells or trace amounts of RNA. With the use of this kit, it is possible
to prepare cDNA from full-length total RNA.
This kit uses Reverse Transcription with Random Displacement Amplification(RT-RamDA™); see method reference (1). RT-RamDA™ is a novel cDNA amplification
method that utilizes the strand displacement activity of reverse transcriptase, and this kit
can detect not only poly(A) RNA but also non-poly(A) RNA with high sensitivity. For
this reason, the RT-RamDA™ method is characterized by being able to detect more genes
than conventional technology.
This kit does not include library preparation reagents or magnetic beads.

Process Workflow

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Features
1. cDNA can be prepared from a single cell or small amount of input RNA
1–100 cells or 10 pg–1 ng total RNA
2. Full-length cDNA can be analyzed
cDNA that covers the entire length of the target RNA of 10 kb or more can be prepared.
3. Various RNAs can be detected, and the number of genes detectable is higher
than that with conventional technology.
◇ Identification of isoforms and alternative splicing◇ Detection of poly(A) RNA and non-poly(A) RNA (histone RNA and lncRNA)◇ Detection of nuclear RNA (pre-mRNA and lncRNA)[ 2 ] Components

The kits include the following reagents that can be used for 96 (RMD-101) and 24(RMD-101T) reactions. All reagents should be stored at −20°C.
Important: Do not store any solutions once mixed. All reagent cocktails should be
prepared shortly before use.

RamDA-seq™ Single Cell Kits (Cat. No. RMD-101 and RMD-101T)①Lysis Buffer②Lysis Enhancer③RNase Inhibitor④Nuclease-free water⑤RT-RamDA™ Buffer⑥RT-RamDA™ Enzyme Mix⑦RT-RamDA™ Primer Mix⑧gDNA Remover⑨2nd strand Synthesis Buffer⑩2nd strand Synthesis Enzyme⑪2nd strand Synthesis Primer Mix

RMD-101480 L108 L22 L
960 L240 L
54 L
54 L
54 L
330 L55 L
275 LRMD-101T
120 L27 L
6 L
240 L
60 L
14 L
14 L
14 L
83 L
14 L
69 L

NSR Primer Set for human samples (Cat. No. NSR-101)
1st NSR Primer Mix for human
2nd NSR Primer Mix for human54 L275 LNSR Primer Set for mouse samples (Cat. No. NSR-102)
1st NSR Primer Mix for mouse
2nd NSR Primer Mix for mouse

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Required materials not included· Thermocycler· Library preparation reagents
The RamDA-seq™ Single Cell Kits are specifically for use with the Illumina
Nextera™ XT DNA Sample Kit.
Nextera™ XT DNA Sample Kit (24 Samples), Cat. no. FC-131-1024
Nextera™ XT DNA Sample Kit (96 Samples), Cat. no. FC-131-1096· SPRI (Solid Phase Reversible Immobilization) paramagnetic beads
Agencourt™ AMPure™ XP Beads (Beckman Coulter, Cat. no. A63880 or A63881)· TE Buffer pH 8.0 (10 mM Tris-HCl,,1mM EDTA)· Magnetic rack/stand for magnetic bead separation· 80% Ethanol (freshly prepared)[ 3 ] Protocol
This kit can be used with cells or purified total RNA. Start with Section 1A when using
FACS to obtain the cells, Section 1B when using non-FACS cells, and Section 1C whenusing purified total RNA. Then proceed with the denaturation in Section 2.
SampleCells

Cell lysis or RNA Dilution
Section 1A
Section 1B
Section 1C

FACS
Non-FACS

Purified RNA
1.

Denaturation
Continue with
Section 2 for all
samples.

Cell lysis or RNA dilution

A. When acquiring cells with FACS
(1) Prepare a cocktail of the cell lysis reagents in a single tube using the volumes listed in
the table below. The volumes used will need to be scaled depending on the number ofreactions you wish to perform.
1 Reaction(μL)①Lysis Buffer②Lysis Enhancer③RNase Inhibitor④Nuclease-free water
Total

2
0.45
0.05
0.5
3

20 Reactions
(μL)*
44
9.9
1.1
11
66

100 Reactions
(μL)*
220
49.5
5.5
55
330

*An extra 10% in volume has been added here to account for pipetting error.

(2) Dispense 3 μL of cell lysis cocktail per well into a 96-well plate or 8-tube strip.
Dispense on ice, and immediately after dispensing, seal with a qPCR seal orthermocompression seal.
(3) Store the plate on ice or at 4°C until sorting, and perform cell sorting according to the
FACS user manual and the parameters recommended by the manufacturer.
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www.toyobo.co.jp/e/bio(4) After sorting, seal the plate or tubes and centrifuge briefly to collect the solution at thebottom of the wells/tubes.
(5) Proceed immediately to the next step or store the samples at −80℃.

B. Cell acquisition using methods other than FACS
When obtaining the cells by manual picking, etc., the volume of the cell sample shouldbe 0.5 μL or less.
(1) Prepare the reagents necessary for cell lysis in one tube per reaction using thevolumes listed in the table below, and then add the cell samples.
If the volume of the cell sample is less than 0.5 μL, adjust the amount of water added
when preparing the cell lysis reaction.

①Lysis Buffer②Lysis Enhancer③RNase Inhibitor
Sample④Nuclease-free water
Total

1 Reaction (μL)
2
0.45
0.05
~0.5
X (adjust as needed based on
sample volume)
3

Example: When the cell sample volume is 0.5 μL
1 Reaction (μL)
①Lysis Buffer
2②Lysis Enhancer
0.45③RNase Inhibitor
0.05
Sample0.5
Total
3(2) Dispense 3 μL of each cell lysis reaction per well into a 96-well plate or 8-tube strip.
Dispense on ice, and immediately after dispensing, seal with a qPCR seal orthermocompression seal.
(3) Seal the plate or tubes and centrifuge briefly to collect the solution at the bottom of
the wells/tubes.
(4) Proceed immediately to the next step or store the samples at −80℃.

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C. When using purified total RNA(1) Prepare the reagent cocktail using the volumes listed in the table below. The
volumes used will need to be scaled depending on the number of reactions you wish to
perform. For example:
Preparation of cell lysis cocktail
100 Reactions (μL)*①Lysis Buffer
220②Lysis Enhancer
49.5③RNase Inhibitor
5.5④Nuclease-free water
55
Total
330
*An extra 10% in volume has been added here to account for pipetting error.

RNA dilution to obtain 1 ng input(μL)
E.g., 10 ng/μL total RNA
1
Nuclease-free water
29
Total
30
Use 3 μL of the diluted 333pg/μL total RNA solution per reaction

RNA dilution to obtain 10 pg input(μL)
E.g., 333 pg/μL total RNA
1
Nuclease-free water
99
Total
100
Use 3 μL of the diluted 3.3 pg/μL total RNA solution per reaction.
(2) Dispense 3 μL of the reagent cocktail per well into a 96-well plate or 8-strip tube
(3) After dispensing, seal and centrifuge briefly to collect the solutions at the bottom of
the wells/tubes.
(4) Proceed immediately to the next step or store the samples at −80℃.

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2． Denaturation(1) Centrifuge the sample plate or tubes at 4℃ and incubate as described in the tablebelow.
Note: When using frozen samples, thaw the samples first at 4°C before heat denaturationand then proceed to heat denaturation after brief centrifugation.

Step
Denature
Hold

Temperature
70℃
4℃

Time
1.5 min.
∞3． Digestion of genomic DNA(1) Prepare a cocktail of the reagents necessary for genomic DNA removal using thevolumes listed in the table below. The volumes used will need to be scaled depending on
the number of reactions you wish to perform.
Note: When using control RNA such as ERCC RNA, adjust the volume of nuclease-freewater added.
1 Reaction(μL)⑤RT-RamDA™ Buffer⑧gDNA Remover④Nuclease-free water
Total

0.3
0.45
2.25
3

20 Reactions
(μL)*
6.6
9.9
49.5
66

100 Reactions
(μL)*
33
49.5
247.5
330

*An extra 10% in volume has been added here to account for pipetting error.

(2) Add 3 μL of the genomic DNA removal cocktail per well to the 96-well plate or
8-tube strip that contains the heat-denatured samples from the previous step. Centrifuge
briefly and incubate at the following temperatures.
Step

Temperature

Genomic DNA digestion
Hold

30℃
4℃

Time
5 min.
∞(3) Proceed immediately to the next step after this reaction.

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4. cDNA synthesis and amplification reaction (RT-RamDA™ reaction)(1) Prepare a cocktail of the necessary reagents for the RT-RamDA™ reaction in a single
tube using the volumes listed in the table below. The volumes used will need to be scaled
depending on the number of reactions you wish to perform.
Note: When using the NSR Primer, use the 1st NSR Primer Mix for human samples or the
1st NSR Primer Mix for mouse samples instead of the RT-RamDA™ Primer Mix.

⑤RT-RamDA™ Buffer⑥RT-RamDA™ Enzyme Mix⑦RT-RamDA™ Primer Mix④Nuclease-free water
Total

1 Reaction
(μL)
1.5
0.45
0.45
0.6
3

20 Reactions
(μL)*
33
9.9
9.9
13.2
66

100 Reactions
(μL)*
165
49.5
49.5
66
330

*An extra 10% in volume has been added here to account for pipetting error.

(2) Add 3 μL of the RT-RamDA™ cocktail solution per well to the 96-well plate or
8-tube strips in which the genome removal reactions were performed. After brief
centrifugation, incubate the plate/tubes at the following temperatures.
Step
Priming 1

Temperature
25℃

Time
10 min.

Priming 2

30℃

10 min.

Reverse transcription andamplification

37℃

30 min.

Reverse transcription 2

50℃

5 min.

Inactivation

98℃

5 min.

Hold

4℃

∞

(3) Proceed immediately to the next step after the RT-RamDA™ reaction or store at−20°C to −30°C.

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5. 2nd strand synthesis reaction(1) Prepare the reagents necessary for the 2nd strand synthesis reaction in a tube using thevolumes listed in the table below. The volumes used will need to be scaled depending on
the number of reactions you wish to perform.
Note: When using the NSR Primer, use the 2nd NSR Primer Mix for human samples andthe 2nd NSR Primer Mix for mouse samples instead of the 2 nd strand Synthesis Primer
Mix.
1 Reaction(μL)⑨2nd strand Synthesis Buffer⑩2nd strand Synthesis Enzyme⑪2nd strand Synthesis Primer Mix
Total

3
0.5
2.5
6

20 Reactions(μL)*

100 Reactions
(μL)*

63
10.5
52.5
126

315
52.5
262.5
630

*An extra 5% in volume has been added here to account for pipetting error.
(2) Add 6 μL of the 2nd strand synthesis cocktail per well of the 96-well plate or 8-tube
strips that contains the RT-RamDA™ reactions and incubate at the following
temperatures after brief centrifugation.

Step
Second-strand synthesis
Denaturation
Hold

Temperature
16℃
70℃
4℃

Time
60 min.
10 min.
∞(3) Proceed immediately to the next step after the 2nd strand synthesis reaction or store
the reactions at −20°C to −30°C.

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6. Library preparation
Important: The library preparation protocol for use with this kit now recommends using
1/4 of capacity typically recommended by Illumina for use with their Nextera™ XTDNA Library Prep Kit. Please note that this change is included in the following
protocol and is only applicable to this kit.
Please refer to the Illumina Nextera™ XT DNA Library Prep Kit manual for an
in-depth description of the library preparation procedure.
A. Purification of double-stranded cDNA
(1) Make 1/4 diluted AMPure XPbeads before the purification step.When diluting,centrifuge AMPure XP beads and use the supernatant as diluent.
Example) How to make 1/4 diluted AMPure XP beads for 8 samples
a. Place 1 × AMPure XP Beads 220 µL on a magnetic stand, and when the magnetic
beads are completely absorbed, use the supernatant as a diluent.
b. Mix 180 μL of the supernatant prepared in step a with 60 μL of 1 × AMPure XP beads
and use as 1/4 diluted AMPure XP beads.
(2) Add 27 μL of 1/4 diluted AMPure XP beads to 15 μL of double-stranded cDNA
solution. After that, centrifuge, at 2,000 rpm for 2 minutes at 25 ° C, and let stand for
5 minutes.
(3) The reaction tubes or 96-well plate is placed on the magnetic stand to allow thesolutions to become clear. Remove the supernatant by careful pipetting.
(4) Add 150 μL of 80% ethanol per well/tube, mix briefly by pipetting the beadsuspension up and down five times, and incubate the solutions for 30 sec at room
temperature.
(5) Place the tubes or plate back onto the magnetic stand and wait until the solutions
become clear. Remove the ethanol by careful pipetting.
(6) Add 150 μL of 80% ethanol per well/tube, mix briefly by pipetting five times, andincubate the solutions for 30 sec at room temperature.
(7) Place the tubes or plate back onto the magnetic stand and wait until the solutions
become clear. Remove the ethanol by careful pipetting.

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www.toyobo.co.jp/e/bio(8) To remove any residual ethanol, leave the tubes or plate open on the magnetic stand atroom temperature to dry for up to 5 min. or until the magnetic beads begin to visibly
crack (if any ethanol remains, the subsequent reactions may not be successful). Adjust
the drying time according to the ambient temperature and humidity in your laboratory.
(9) Prepare the reagents necessary for elution in a separate tube while waiting for thebeads to dry, using the volumes listed in the table below. The volumes used will need
to be scaled depending on the number of reactions you wish to perform.

1 Reaction(μL)
Tagment DNA Buffer(Nextera™ XT DNA Library Prep Kit)
④Nuclease-free water
Total

100 Reactions
(μL)*
2.5

270

1.25
3.75

135
405

*An extra 8% in volume has been added here to account for pipetting error.
(10) Add 3.75 μL of the elution cocktail to each reaction in the 96-well plate or 8-tube
strips and briefly centrifuge.
(11) Vortex until the beads are completely dispersed, incubate at 25°C for 5 min., andbriefly centrifuge to collect the suspension at the bottom of the wells/tubes.
(12) Place the tube or plate on the magnetic stand for 5 minutes or until the solutions
have cleared.
(13) Transfer 3.75 μL of the clear supernatant to new tubes or a plate and proceed to thenext step.
B. Tagmentation (Nextera™ XT DNA library preparation)(1) Add 1.25 μL of Amplicon Tagment Buffer (Nextera™ kit) to each of the 3.75 μL
solutions containing purified double-stranded cDNA. Centrifuge briefly and incubate
at the following temperatures.
Step
Tagmentation
Hold

Temperature
55℃
10℃

Time
5 min.
∞

C. Neutralization (Nextera™ XT DNA library preparation)(1) Add 1.25 μL per well of Neutralization Buffer to the tagged double-stranded cDNA
prepared in the previous step. Centrifuge briefly, incubate at 25°C and 2,000 rpm for 1
min., and then let the reactions stand for 5 min.

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D. Library enrichment (Nextera™ XT DNA library preparation)(1) Add 3.75 μL of NPM PCR Master Mix to each reaction containing the neutralized
double-stranded cDNA and briefly centrifuge. Then, add 1.25 μL each of the index
primers S5XX and S7XX to each well and briefly centrifuge.
(2) After centrifugation, perform PCR with the following cycling conditions.
Step

Temperature

Time

Anneal72℃

3 min.

No. ofcycles1

Denature

95℃

30 sec.

1

PCR

Extension

95℃
55℃
72℃
72℃

10 sec.
30 sec.
30 sec.
5 min.

Hold

4℃

∞

14*
1
1

*Change the number of PCR cycles according to the table below.
Estimated amount of
total RNA per cell/total
RNA amountTypical number of
PCR Cycles>10 pg14

5–10 pg

14–15*If the amount of input RNA is less than 5 pg, increase the number of PCR cycles.
Note: If the library yield is too high, the number of PCR cycles can be reduced.
E. Library purification (Nextera™ XT DNA library preparation)(1) Add 15 μL of 1× AMPure XP beads to 12.5 μL of each PCR reaction. Centrifuge
briefly, incubate for 2 min. at 25°C and 2,000 rpm, and then let the reactions stand for
5 min.
(2) Place the tubes or plate on a magnetic stand and allow the solutions to clear. Remove
the supernatants by careful pipetting.
(3) While still on the magnetic stand, add 150 μL of 80% ethanol to each well/tube, mix
briefly by pipetting five times, and incubate at room temperature for 30 sec.
(4) Remove the ethanol by careful pipetting.
(5) With the tubes or plate on the magnetic stand, add 150 μL of 80% ethanol per sample,
mix briefly by pipetting five times, and incubate at room temperature for 30 sec.

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www.toyobo.co.jp/e/bio(6) Remove the ethanol completely by careful pipetting.
(7) Leave the tubes or plate on the magnetic stand and allow the beads to dry at room
temperature for 2 min. or until the magnetic beads begin to crack (if any ethanol
remains, the subsequent reactions may not be successful). Adjust the drying time
according to the ambient temperature and humidity of your laboratory.
(8) Add 24 μL of TE Buffer per well/tube and briefly centrifuge.
(9) Vortex until the beads are completely dispersed, incubate at 25°C for 2 min. and thenbriefly centrifuge.
(10) Place the tubes or plate on the magnetic stand and allow to stand for 2 min. until thesolutions clear.
(11) Transfer 24 μL of the clear supernatant to a new plate or tubes and proceed to the QC
step or store at −20°C.

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7. Library QC
Library quantification
Library quantification can be performed using 7. Library QC
Use the Toyobo GenNext™ NGS Library Quantification Kit (Cat. No. NLQ-101) orequivalent. This kit allows the specific and accurate quantification of libraries bearing P5
and P7 adapters and can be applied to flow cell amplification.
Library quality control
Assess the quality of the library using a capillary electrophoresis device such as an
Agilent Bioanalyzer or equivalent.
Library distribution example
When using the GenNext™ RamDA-seq™ Single Cell Kit together with the Nextera™
XT DNA Library Preparation Kit (14 cycles), and when preparing a library from mouse
ES cells for, on average, a single cell per reaction, the library distribution was confirmed
to be in the range of 100–600 bp as shown in the figure below. An eluted library
concentration of 10–20 nM (with an elution volume of 24 μL in TE buffer) was obtained.

8. Sequence QC
It is recommended to check the coverage uniformity before analyzing the sequence data.
With this kit, it is possible to obtain uniform coverage as shown below.

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www.toyobo.co.jp/e/bio[ 4 ] Application data
1. Comparison of the number of detected transcripts and the alignment ratio within eachregion<Method> Using next generation sequencing data, cDNA and double-stranded DNA wereprepared using either the GenNextTM RamDA-seq™ Single Cell Kit (Cat. No. RMD-101)
or a Company A kit, with a sample input of 10 pg of total RNA extracted from mouse ES
cells. The GenNextTM RamDA-seq™ Single Cell Kit uses the NSR Primer Set for mouse
samples (NSR-102). PCR for cDNA amplification, which is not required with the
GenNextTM RamDA-seq™ Single Cell Kit, was performed for 18 cycles with theCompany A kit. The libraries were then prepared with the Nextera™ XT DNA Library
Preparation Kit and sequenced with the Illumina MiSeq instrument.
<Result>

Thus, the GenNextTM RamDA-seq™ Single Cell Kit was shown to detect approximately
5,000 more genes than the Company A kit.
2. Comparison of the full-length coverage of long RNA molecules<Method> Analysis was performed on the sequence data obtained with the method
described for Example 1.
<Result>

The GenNextTM RamDA-seq™ Single Cell Kit can detect lncRNAs such as Neat1 andMalat1, which were difficult to capture with the Company A kit over their entire length.

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www.toyobo.co.jp/e/bio[ 5 ] Troubleshooting
Symptom

Cause

Low cDNA yield

RNA is degraded· Check that the RNA is not degraded.
· Check that RNase has not contaminated the tips or
tubes used.
· Perform all cell lysis and RNA dilution steps on ice.

Low library yield

Ethanol remaining after
purification· If ethanol remains among the magnetic beads after
washing with ethanol, subsequent enzyme reactions may
be hindered. Make sure that the magnetic beads are dry.
· If the humidity in the laboratory is high, it may bedifficult to dry the beads. It is recommended that thehumidity in the work area be 55% or less.

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www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

Solution

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

15

www.toyobo.co.jp/e/bio[ 6 ] Related products
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cDNA prep kit for qPCR

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GenNext™ NGS Library Quantification Kit
For the quantification of NGS libraries prepared with

No. of reactions
96
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Catalog No.
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Reference (1)Tetsutaro Hayashi*, Haruka Ozaki*, Yohei Sasagawa, Mana
Umeda, Hiroki Danno and Itoshi Nikaido. Single-cell full-length
total RNA sequencing uncovers dynamics of recursive splicing andenhancer RNAs. Nature Communications. 2018.

JAPANTOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

16