CATS Total RNA-seq Kit (with rRNA depletion) v2 x24

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

		Total time	Hands-on-time
CATS total RNA-seq rRNA depletion (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.

• 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.