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CATS Small RNA-seq Kit x24

Catalog Number
24 rxns
Other format

D-Plex Small RNA-seq library prep kit for Illumina

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


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


    STEP 1
    ssRNA end-repair
    and polyA tailing

    75 min 10 min

    STEP 2
    RT and template switch

    150 min 10 min


    STEP 3
    PCR amplification
    of DNA library

    30 min 5 min


    (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.

  • 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.
  •  Documents
    CATS Small RNA-seq kit MANUAL
    CATS Small RNA sequencing kit for Illumina
    Technical note CATS small RNA sequencing APPLICATION NOTE
    The Diagenode CATS Small RNA-seq kit generates ready-to-sequence libraries on the Illumina platfo...
    Diagenode trimming tools for CATS library preparation [read1]
    Due to special mechanisms in creating CATS libraries such as template switching and artificial po...
    Diagenode trimming tools for CATS library preparation MANUAL
    This document describes the usage of the Diagenode trimming tools for CATS library preparation, w...
    Material Safety Data Sheet CATS small RNA-seq Kit DATASHEET
  •  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!

    Repeat RNAs associate with replication forks and post-replicative DNA.
    Gylling HM, Gonzalez-Aguilera C, Smith MA, Kaczorowski DC, Groth A, Lund AH
    Non-coding RNA has a proven ability to direct and regulate chromatin modifications by acting as scaffolds between DNA and histone-modifying complexes. However, it is unknown if ncRNA plays any role in DNA replication and epigenome maintenance, including histone eviction and re-instalment of histone-modifications aft...

    Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.
    Herbert ZT, Thimmapuram J, Xie S, Kershner JP, Kolling FW, Ringelberg CS, LeClerc A, Alekseyev YO, Fan J, Podnar JW, Stevenson HS, Sommerville G, Gupta S, Berkeley M, Koeman J, Perera A, Scott AR, Grenier JK, Malik J, Ashton JM, Pivarski KL, Wang X, Kuffe
    Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been chall...

    Circulating miRNA analysis for cancer diagnostics and therapy.
    Valihrach L, Androvic P, Kubista M
    Successful treatment of cancer depends on early diagnosis and effective monitoring of patients' response to therapy. Traditional tools based on tumor biopsies lack the sensitivity and specificity to capture cancer development in its early phases and are not applicable for continuous monitoring. To overcome these bar...

    The sncRNA Zoo: a repository for circulating small noncoding RNAs in animals.
    Fehlmann T, Backes C, Pirritano M, Laufer T, Galata V, Kern F, Kahraman M, Gasparoni G, Ludwig N, Lenhof HP, Gregersen HA, Francke R, Meese E, Simon M, Keller A
    The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expres...

    MicroRNAs and Transplantation.
    Khan Z, Suthanthiran M, Muthukumar T
    miRNAs, ∼20 to 22 nucleotide single-stranded RNA species that play a pivotal role in the regulation of protein-coding genes, are emerging as robust biomarkers for assessing allograft status. Herein, the authors briefly review the biogenesis and function of the miRNAs and provide an overview of the tools to quant...

    NGS analysis of total small non coding RNAs from low input RNA from dried blood sampling
    Marcello Pirritano, Tobias Fehlmann, Thomas Laufer, Nicole Ludwig, Gilles Gasparoni, Yongping Li, Eckart Meese, Andreas Keller, and Martin Simon
    Circulating miRNAs are favored for biomarker candidates as they can reflect tissue specific miRNA dysregulation in disease contexts. Moreover, they have additional advantages that they can be monitored in a minimal invasive manner. Blood-borne miRNAs are therefore currently characterized to identify, describe and va...

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