This kit contains and rRNA depletion module that depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA, 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA samples. This product is suitable for both intact and degraded RNA preparations (e.g. FFPE tissue).
- 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 total RNA-seq rRNA (-) library preparation workflow
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Total time |
Hands-on-time |
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CATS total RNA-seq
rRNA depletion (module included)
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60-120 min |
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(1)
SAFE STORAGE
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STEP 1
ssRNA end-repair
and polyA tailing
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75 min |
10 min |
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(2)
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STEP 2
RT and template switch
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150 min |
10 min |
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(3)
SAFE STORAGE
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STEP 3
PCR amplification
of DNA library
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30 min |
5 min |
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(4)
SAFE STORAGE
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(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.
