Diagenode

CATS mRNA-seq Kit (with polyA selection) v2 x24

CATS
Catalog Number
Format
Price
C05010043
24 rxns
$1,450.00
Other format

Diagenode’s CATS mRNA-seq Kit (with polyA selection) 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. The poly (A) + RNA module in this kit is designed for the isolation of poly (A) + RNA transcripts from total RNA for RNA library preparation and sequencing. 

  • Diverse transcripts detection
  • Excellent reproducibility with ultra low inputs down to 100 pg
  • Great performance on challenging samples
  • 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 100 picograms of polyA selected material 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.

Broad overview of the transcriptome

Biotypes* represented in the libraries CATS v2 mRNA-seq Kit vs NEBNext Ultra I directional RNA-seq Kit. TPM ≥2.
Input: 1 ng, 10 ng poly(A) RNA from human universal total RNA (Agilent, 740000). * < 0.04% of total detected transcripts at this threshold is rRNA

Retained complexity at low inputs

Biotypes* represented in the libraries CATS v2 mRNA-seq Kit. TPM ≥2.
Input: 1 ng, 10 ng poly(A) RNA from human universal total RNA (Agilent, 740000). * < 0.04% of total detected transcripts at this threshold is rRNA

Uniquely detected transcripts

All detected transcripts in CATS v2 mRNA-seq Kit vs NEBNext Ultra I directional library preparation Kit. No threshold has been set for the analysis.
Input: 1 ng poly(A) RNA from human universal total RNA (Agilent, 740000). 36,245 transcripts found in both libraries.

Consistency and wide representation of the transcriptome

Comparison on the number of coding transcripts detected with CATS v2 mRNA-seq Kit vs SMARTer stranded RNA-seq library preparation kit. TPM ≥2.
Input: 1 ng poly(A) RNA from human universal total RNA (Agilent, 740000). 21,333 transcripts found in both libraries.

High-complexity at low inputs

Mapped reads distribution for CATS v2 mRNA-seq Kit vs NEBNext Ultra I directional RNA-seq Kit.
Input: 1 ng, 10 ng poly(A) RNA from human universal total RNA (Agilent, 740000).

High correlation in transcripts detection across ultra low inputs

Transcripts detected in 1 ng vs 0.1 ng samples of CATS v2 mRNA-seq libraries. TPM≥2.
Input: 1 ng, 0.1 ng poly(A) RNA from human universal total RNA (Agilent, 740000).

Very reproducible library preparation method

Transcripts detected in two technical replicates of CATS v2 mRNA-seq libraries. TPM≥2
Input: 1 ng poly(A) RNA from human universal total RNA (Agilent, 740000).

Few PCR cycles, less amplification bias

BioAnalyzer® electropherogram of two technical replicates of CATS v2 mRNA-seq libraries.
Input: 1 ng rRNA(-) RNA from human universal total RNA (Agilent, 740000).

CATS saves time



TESTIMONIAL

I like the Diagenode CATS RNA-seq kit for a number of reasons. 
The kit is approachable for any research user. The things I care about are time, reproducibility, and ease. In my hands, the CATS mRNA-seq kit has generated highly reproducible libraries from very low input RNA that goes into polyA selection. This has significantly decreased the amount of time I spend doing library preps, enabling me to further engage with my research.

Jordan Lewandowski, PhD; HSCRB // Harvard
  • Characteristics

    This kit contains a poly (A) + RNA module for the isolation of poly (A) + RNA transcripts from total RNA for RNA library preparation and sequencing. The module is designed for using 1 µm paramagnetic beads coupled with oligo d (T) 25 chains. This structure is then used to capture RNA bearing a poly (A) queue using a gentle, magnetic isolation for intact, full-length mRNA.

    • Ligation-free  assay - increased efficiency and minimum bias
    • Low inputs down to 100 picograms
    • 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 mRNA-seq library preparation workflow

    Total time Hands-on-time

    CATS mRNA-seq
    polyA selection (module included)

    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 mRNA-seq kit (with polyA selection) MANUAL
    CATS mRNA-seq kit (with polyA selection) manual
    Download
    CATS paired-end sequencing primer (for read2) DATASHEET
    This primer has been specifically designed to be used in paired-end sequencing with CATS RNA-seq ...
    Download
    Diagenode trimming tools for CATS RNA-seq [read1]
    Due to special mechanisms in creating CATS libraries such as template switching and artificial po...
    Download
    Diagenode trimming tools for CATS RNA-seq [read2]
    Due to special mechanisms in creating CATS libraries such as template switching and artificial po...
    Download
    Diagenode trimming tools for CATS RNA-seq MANUAL
    This document describes the usage of the Diagenode trimming tools for CATS RNA-seq, which consist...
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: CATS mRNA-seq Kit (with polyA selection) v2 x24 (Diagenode Cat# C05010043). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

  • FAQs
    • In the CATS mRNA-seq kit, will the natural polyA tails undergo the polyadenylation step?
      Yes. The RNA fragments containing the natural polyA tail will be tailed as well during the polynucleotide tailing step.
    • What fragmentation should I carry if my sample has a RIN of 2-3?
      For heavily degraded RNA samples with a RIN of < 3, no fragmentation step is recommended to start the library preparation. However, the fragmentation buffer still needs to be added in the sample but no heat should be applied (please skip high temperatures in the “fragmentation step” in manuals).
    • How long is the CATS RNA-seq library preparation protocol?
      For the RNA-seq library preparation itself, takes 4h30-5h to complete for a trained user. This does not include the sample preparation (e.g. polyA selection, rRNA depletion or small RNA enrichment) and the QC of the library. Each of those two steps take additional approximate 1h.
    • I am working with mid- to low-quality RNA. Are there any recommendations?
      We recommend to run a BioAnalyzer (or equivalent) electrophoresis to get the fragmentation profile of the RNA. Then you should decide in collaboration with the Diagenode technical support customer.support@diagenode.com about the best fragmentation time to use for your RNA samples.
    • What would be the lowest possible limit for the input for rRNA depletion and polyA selection modules in CATS kits?
      It is possible to use lower amount of starting material than mentioned in manuals. To get more information about this, contact Diagenode technical support.

    • Before mapping, CATS RNA-seq data requires a specific trimming procedure described in all manuals. Please consult also a user guide “Diagenode trimming tools for CATS RNA-seq”.
    • 7) Is CATS compatible with directional mRNA-seq?
      Yes. CATS RNA-seq library preparation retains strand specificity which is also known as directional library preparation.
    • 8) Which RIN do my RNA samples need to have to work with CATS RNA-seq kits?
      We suggest using high quality RNA (RIN>8) but degraded RNA can be also used. In case of degraded RNA, the fragmentation time must be adjusted. In the protocol we advise 7 min at 94°C for fragmenting high quality RNA (RIN>8). If the RNA is severely degraded like RNA derived from FFPE tissue (RIN 2-3), the fragmentation may be skipped.
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