- 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 RNA-seq library preparation workflow
|
|
|
Total time |
Hands-on-time |
|
|
(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.
