Description

GenNext RamDA-seq™ Single Cell Kit (TYB-RMD-101, TYB-RMD-101T) is a kit using RT-RamDA™ (Reverse Transcription with Random Displacement Amplification) method for preparing cDNA for NGS analysis from single-cell and Ultra-low amount RNA. RT-RamDA™ is a novel cDNA amplification method utilizing the strand displacement activity of reverse-transcriptase. GenNext RamDA-seq™ Single Cell Kit can detect not only poly(A) RNA but also non-poly(A) with high sensitivity by using random primer or NSR primer^*, allowing the detection of many genes compared with the conventional method.

^* RamDA-seq™ and RT-RamDA™ are a trademark of RIKEN, Institute of Physical and Chemical Research
^* NSR primer is abbreviation for Not so random primer. NSR primer is designed random primer to avoid synthesizing cDNA from the rRNAs by removing 6-mers that exactly match the rRNA sequences from N6 random primers. In order to perform more efficient NGS analysis, it is necessary to reduce the rRNA contamination rate. As a countermeasure, we also offer Not so random primers for humans and mice sold separately.

Features

Explore the RamDA-seq™ Kit Family

Both kits use the RIKEN-developed RT-RamDA™ method for full-length total RNA-seq from 1-100 cells or 10 pg-1 ng total RNA. Choose the kit by the output your workflow needs.

Components

Select Kit Size & NSR Primer Sets

Kit 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.

*Do not store mixed solution. To use this kit, we recommend using NSR primers.

Storage Conditions

Application Data

Example 1. Comparison of the number of detected transcripts and the alignment ratio within each region

Using next generation sequencing data, cDNA and double-stranded DNA were prepared using either the GenNext™ RamDA-seq™ Single Cell Kit (Code No. TYB-RMD-101) or a Company A kit, with a sample input of 10 pg of total RNA extracted from mouse ES cells. The GenNext™ RamDA-seq™ Single Cell Kit uses the NSR Primer Set for mouse (Code No. TYB-NSR-102). PCR for cDNA amplification, which is not required with the GenNext™ RamDA-seq™ Single Cell Kit, was performed for 18 cycles with the Company A kit. The libraries were then prepared with the Nextera™ XT DNA Library Preparation Kit and sequenced with the Illumina MiSeq instrument. Thus, the GenNext™ RamDA-seq™ Single Cell Kit was shown to detect approximately 5,000 more genes than the Company A kit.

Example 2. Comparison of the full-length coverage of long RNA molecules

Analysis was performed on the sequence data obtained with the method described for Example 1. The GenNext™ RamDA-seq™ Single Cell Kit can detect lncRNAs such as Neat1 and Malat1, which were difficult to capture with the Company A kit over their entire length.

Example 3. Comparison of the rate of rRNA contamination using random Primers or NSR Primers

Using the RT-RamDA™ Primer Mix (random Primers) included with the GenNext™ RamDA-seq™ Single Cell Kit [Code No. RMD-101] or the separately sold NSR Primer Set for mouse [Code No. NSR-102], cDNA and double-stranded DNA were synthesized from total RNA (10pg, 1ng) extracted from NIH3T3 cells. Libraries were prepared using the Nextera™ XT DNA Library Preparation Kit and Sequencing was performed on an Illumina™ MiSeq™ instrument.

For both 10pg and 1ng of Total RNA, NSR Primers resulted in the lower rate of rRNA contamination compared to random primers.

Example 4. RNA-seq analysis from various RNA input amounts (2 pg to 6.25 ng)

Using RNA extracted from HeLa cells, libraries were prepared with input amounts ranging from 2 pg to 6.25 ng. For cDNA synthesis, the NSR Primer Set for human [Code No. NSR-101] was used, and all library enrichments were performed with 15 cycles. Sequencing was performed on an Illumina™ MiSeq™ instrument, and NGS analysis was performed using basespace (Illumina) RNA-Seq Alignment (ver. 2.0.2) or RamDAQ (ver.1.9).

The number of detected transcripts and genes was low at 2 pg because the input amount was low to begin with. The number of detected transcripts and genes was high at input amounts other than 2 pg, and the coverage was unbiased at all input amounts.

Principle of the RT-RamDA™ Method

RT-RamDA™ is an abbreviation for "Reverse Transcription with Random Displacement Amplification" developed by the Bioinformatics Research and Development Team, RIKEN Center for Biosystems Science and Technology. It is a new cDNA amplification method that applies the strand substitution activity of reverse transcriptase, and it is possible to prepare cDNA not only from poly(A) RNA but also from non-poly(A)-derived RNA for using random primer.

Unlike conventional method using Oligo-dT primer, cDNA for full-length total RNA-seq can be synthesized by RT-RamDA™ method.

In addition, because cDNA is amplified at the same time as it is synthesized, RT-RamDA™ method does not require amplification adaptors and PCR amplification step, and it reduces the biases caused by PCR amplification.

1. Hayashi T. et al. Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs. Nature Communications. 9: 619 (2018)

Workflow

Publications Using GenNext™ RamDA-seq™ Single Cell Kit

1. Yoshimatsu, S. et al., Non-viral Induction of Transgene-free iPSCs from Somatic Fibroblasts of Multiple Mammalian Species. Stem Cell Reports, Volume 16:754-770(2021)
2. Hasegawa, N. et al., Highly sensitive fusion detection using plasma cell-free RNA in non-small-cell lung cancers. Cancer Sci. 112(10):4393-4403(2021)
3. Oka, Y. et al., A novel sorting signal for RNA packaging into small extracellular vesicles. Scientific Reports 13, Article number: 17436 (2023)
4. Usuda, M. et al., Inhibitory Effect of Adsorption of Streptococcus mutans onto Scallop-Derived Hydroxyapatite. Int. J. Mol. Sci. 24,11371 (2023)

Publications

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Bioz AI Summary:

The GenNext RamDA seq Single Cell Kit from Toyobo has been instrumental in a variety of advanced research applications. This kit has enabled high-throughput sequencing and detailed gene expression profile analysis in studies involving gastrointestinal tract samples, S-phase progression, iPSC-derived neurons, and S. mutans under different conditions. It facilitated the preparation of high-quality RNA libraries for single-cell RNA sequencing (scRNA-seq) in tendon cells and lymphatic endothelial cells (LECs), revealing insights into tendon development, metastasis, and cellular heterogeneity in osteoarthritis. The kit's cDNA synthesis capability was crucial for detecting fusion genes in plasma, analyzing viral transcriptomes, and studying the evolution and spread of SARS-CoV-2. Additionally, it supported the analysis of small extracellular vesicles (sEVs) and the role of PIEZO1 in tendon function. The GenNext RamDA seq Single Cell Kit has proven effective in integrating glycan and RNA analysis in single cells, contributing to the comprehensive understanding of gene expression in various biological contexts.

Bioz Signature Insights

Toyobo's GenNext RamDA seq Single Cell Kit has emerged as a pivotal tool in the realm of single-cell RNA sequencing, facilitating groundbreaking research in diverse biological contexts. This innovative kit has been prominently featured in high-impact journals, underscoring its significance in advancing our understanding of gene expression in tendon development, metastasis, and cellular heterogeneity in osteoarthritis. Its robust cDNA synthesis capability has been instrumental in detecting fusion genes, analyzing viral transcriptomes, and studying the evolution and spread of SARS-CoV-2.

Cited Since

2021

Top Journal

Nature communications

Top Journals

Nature communications · IF 15.7
Science translational medicine · IF 14.6
JCI insight · IF 6.1

96/100Bioz Rating

21Citations

4Tables

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