Diagenode

CATS Small RNA-seq Kit x24

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Catalog Number
Format
Price
C05010040
24 rxns
$1,410.00
Other format

Diagenode’s CATS small RNA-seq Kit utilizes the innovative “Capture and Amplification by Tailing and Switching” (CATS), a ligation-free method to produce DNA libraries for next generation sequencing from low input amounts of RNA.

  • Higher small RNA diversity
  • Excellent reproducibility with ultra low inputs down to 10 pg
  • Great performance on challenging samples
  • Straightforward protocol: either gel or magnetic bead-based purification for your choice
  • Read more

    CATS relies on polynucleotide tailing for capturing the 3’-ends of nucleic acids and the template switching ability of MMLV-RT to capture the 5’ ends for cDNA synthesis. This kit allows for generating Illumina compatible ready-to-sequence libraries from RNA inputs as low as 10 picograms in just few hours.

    Libraries retain strand specificity of origin. Unlike competing solutions, our CATS RNA-seq kit exhibits higher library efficiency versus ligation-based methods providing higher complexity from better template capture and minimal bias due to lower amplification requirements.

Unique sncRNAs detected in circulating plasma RNA

sncRNA transcripts detected using the CATS small RNA-seq Kit vs the Competitor’s Kit. The input was 1 ng circulating small RNA in plasma samples. In total, 908 transcripts were found in the CATS small RNA-seq libraries. TPM≥1

PCR cycles: CATS 14x vs Competitor 21x. Final library yield: CATS 27 ng vs Competitor 1.2 ng.

More miRNA transcripts detected from challenging samples

Transcripts detected in miRNA samples of CATS small RNA-seq Kit vs the Competitor’s Kit.

Input: 1 ng circulating small RNA in plasma samples. PCR cycles: CATS 14x vs Competitor 21x. Final library yield: CATS 27 ng vs Competitor 1.2 ng.

Ultra low inputs, excellent reproducibility

Correlation between 2 different operators using CATS small RNA-seq Kit on two different inputs (100 pg vs 1 ng) for ncRNA detected at TPM ≥2.
R2 = 0.86

Consistency across template switching techniques

Comparison of the number of detected small non-coding RNAs CATS small RNA-seq Kit vs SMARTer
Input: 100 pg isolated small RNA. TPM ≥2. Transcripts taken into consideration: miRNA, piRNA, scRNA, scaRNA, snoRNA, snRNA and vaultRNA. 1,217 transcripts found in both libraries at this expression level.

High number of reads mapped

Representation of detected transcripts different biotypes with CATS small RNA-seq Kitvs NEBNext UltraI kit. TPM≥2.
100 ng total RNA, 1 ng small RNA used as input.

Excellent performance on challenging samples

Heatmap clustering of the top 50 differentially expressed miRNAs after library preparation:

  • CATS Small RNA-seq Kit on 1 ng exosomal RNA (Exosome sample 1, 2)
  • CATS RNA-seq Kit on 1 ng total RNA from parent (HEK293T) cells (Cell sample 1, 2).

Prior library preparation, exosomes were isolated with Diagenode exosome capture solutions Exosomes

CATS saves time



TESTIMONIAL

Given the low input range of up to 1 ng of our cell free plasma derived small RNA samples, commercially available kits and various published protocols failed in generating a library suitable for NGS with attempts resulting in high concentrations of adaptor dimer or failed library amplifications.

In light of this, we are more than happy to have tested the CATS small RNA-seq kit by Diagenode. Even with limited input material we got a rich library with absolutely no detectable adaptor dimer, as expected given it is a ligation free method. The kit performed superbly, in contrast to other commercially available kits or published protocols, with less hassle and with a greater ease of use.

Highly recommended for anyone who is struggling with generating a library from low input and challenging samples.

 

Jonatan Darr, PhD, Environmental Epigenetics Group, Institute of Experimental Genetics, Helmholtz Zentrum München, Germany
  • Characteristics
    • Ligation-free  assay - increased efficiency and minimum bias
    • Low inputs down to 10 picograms
    • Higher small RNA diversity
    • User-friendly: reduced hands-on  time with fewer steps than competing methods
    • Libraries ready to sequence in only 5 hours
    • Retaining strand specificity of origin

    CATS small RNA-seq library preparation workflow

    Total time Hands-on-time

    Recommended isolation with
    miRNA easy Mini Kit, Qiagen
    CATS Small RNA Kit

    60-120 min
    (1)

    SAFE STORAGE

    STEP 1
    ssRNA end-repair
    and polyA tailing

    75 min 10 min
    (2)

    STEP 2
    RT and template switch

    150 min 10 min
    (3)

    SAFE STORAGE

    STEP 3
    PCR amplification
    of DNA library

    30 min 5 min
    (4)

    SAFE STORAGE

    (1) RNA: stored at -80°C after purification (safe stop & storage) ; (2) cDNA: stable at 4°C overnight, store at -20°C ; (3) Amplified library: prior purification and QC store at -20°C (safe stop & storage) ; (4) DNA libraries: store at -20°C or -80°C (safe stop & storage)

    Schematic representation of the workflow used by the CATS Small RNA-seq Kit. Single stranded RNAs are first dephosphorylated (end-repaired) and polyadenylated at the 3’-end. Subsequently, a cDNA strand synthesis is performed in the presence of the anchored poly(dT) oligonucleotide containing terminal P7 Illumina® adaptor sequence. When the reverse transcriptase reaches the 5’-end of the RNA it switches the template and continue DNA synthesis over the template-switching oligonucleotide (TSO). The TSO contains three 3’-terminal ribonucleotides X (rX) which facilitate the template switching and carry the terminal P5 Illumina® adaptor sequence. During PCR pre-amplification of the first cDNA strand, Illumina® adapters carrying P5 and P7 terminal sequences (required for clustering on an Illumina® flow cell) as well as index sequences are incorporated into the library. The sum size of the adapters (the size of “empty” library) is 143 bp.

  • Data analysis and interpretation

    Single-click visual informatics and gene expression analysis for CATS RNA-seq kits powered by Genialis™ Expressions software

    Take control of your data with a fully automated data processing pipeline developed and validated for use with Diagenode’s CATS RNA-seq kits. Combined with Genialis’ guided data interpretation workflows in their interactive visual interface, you can:

    • Establish reproducibility across projects. Run your raw data through the CATS-RNA-seq pipeline so your primary data analyses are done the same way across projects.
    • Ensure experiment design quality. Verify consistency between your experimental replicates and identify relevant correlations or differences amongst experimental conditions.
    • Compare expression levels across conditions. Quantify gene abundance in individual samples and investigate differential expression of individual genes, predefined gene sets, and entire transcriptomes.
    • Assess differences in biological activity. Understand how specific experimental conditions affect the regulation of groups of related genes and their corresponding biological functions.

    To learn more about the CATS RNA-seq data analysis workflow, visit genial.is/CATS-workflow.

    See CATS RNA-seq data analysis in action at genial.is/CATS-tutorial-video.

    Request a personal walkthrough of CATS RNA-seq data analysis on Genialis’ Expressions platform at genial.is/CATS-RNA-seq-demo.

  • Applications
    DNA/RNA library preparation
    Most of the major next-generation sequencing platforms require ligation of specific adaptor oligos to fragmented DNA or RNA prior to sequencing After input DNA has been fragmented, it is end-repaired and blunt-ended. The next step is a A-tail... Read more
  • Documents
    CATS Small RNA-seq kit MANUAL
    CATS Small RNA sequencing kit for Illumina
    Download
    Technical note CATS small RNA sequencing APPLICATION NOTE
    The Diagenode CATS Small RNA-seq kit generates ready-to-sequence libraries on the Illumina platfo...
    Download
    Diagenode trimming tools for CATS library preparation [read1]
    Due to special mechanisms in creating CATS libraries such as template switching and artificial po...
    Download
    Diagenode trimming tools for CATS library preparation MANUAL
    This document describes the usage of the Diagenode trimming tools for CATS library preparation, w...
    Download
    Material Safety Data Sheet CATS small RNA-seq Kit DATASHEET
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: CATS Small RNA-seq Kit x24 (Diagenode Cat# C05010040). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

  • FAQs
    • I would like to look at small RNAs of specific sizes (20-100 nt). Would it be possible to do a gel purification instead of purification with AMPure beads?
      We recommend to perform a gel extraction or purification on obtained libraries. Please consult manuals for an established protocol for gel-based purification of your libraries as well as purification with AMPure beads.
    • Will CATS small RNA-seq kit incorporate piRNA during preparation?
      Yes. CATS small RNA-seq incorporate piRNA in prepared libraries.
    • I plan to size select the small RNA libraries with AMPure beads. Can I erform just size selection with AMPure beads straight away without post-PCR clean up?
      The post-PCR clean up is still needed before any further size selection step using AMPure beads in order to remove the PCR primers and the short fragments first.
    • Have we tested CATS small RNA-seq on plasma or serum samples?
      Yes. CATS technology works on RNA extracted from serum and plasma samples. It works very well for circulating RNAs.

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