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
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Total time |
Hands-on-time |
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CATS mRNA-seq
polyA selection (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.
