TSwitch™ 3rd Generation Sequencing Transcriptome Library Construction Kit provides an efficient and convenient method for synthesizing high-quality cDNA from nanogram total RNA based on Template-Switching technology (TSwitch™ technology). TSwitch™ is ideal for samples with limited initial RNA. It can synthesize high-quality cDNA starting from 2 ng total RNA (or very low concentration samples) for various downstream applications, including hybridization probes, virtual northern blots, cDNA sequencing, etc.
The 1st strand cDNA synthesis is initiated by optimized Oligo 1 primer. When the reverse transcription reaches the 5’ end of the ssRNA template, the terminal transferase activity of the reverse transcriptase will add several dC residues at the 3' end of the cDNA. And the annealing between Oligo 2 primer and dC residues added at the 3' end of cDNA will provide an extensible template for the reverse transcription process. Thus the reverse transcriptase will switch the template and complete the synthesis of double-stranded cDNA (TSwitch™ cDNA).
Easy Identification of Source
Different adaptor primers are used for the 5 'and 3' ends, thus the source of reads (whether it is mRNA or cDNA) can be directly judged from the original data.
Compatible with Nanopore
TSwitch™ technology generates high-quality double-stranded cDNA, which meets the requirements of Nanopore.
Shorter Operation Time
The library construction products can be directly connected to the ring sequencing adaptor of PacBio. The two main steps of DNA damage repair and DNA end repair are omitted, thus the operation time is greatly shortened.
Reflecting the Homogeneity of the Transcriptome
The template of this kit is the total RNA of the sample, which reflects the homogeneity of the transcriptome of the sample to the greatest extent.
Saving Sequencing Resources
According to the reading length characteristics of PacBio, the kit adds purification steps to make the initial length of the product 400 bp, greatly saving sequencing resources.
Higher Proportion of Full-Length cDNA
Compared with the cDNA obtained by traditional RACE or library, TSwitch™ RACE has a higher proportion of full-length cDNA.
For Full-Length Transcriptome Sequencing
The operation process can be seamlessly integrated into PacBio and Oxford Nanopore sequencing platforms. After connecting with the adaptors of the corresponding platform and sequencing on the instrument, the full-length transcript of RNA molecules from 5' end to 3' end can be obtained directly. New genes and transcriptional isomers can be accurately found without splicing and assembly, and variable splicing and gene fusion can be identified.
For Virtual Northern Blot
In the case of insufficient mRNA or total RNA, cDNA can be hybridized to analyze the size and expression pattern of transcripts. Virtual northern blot with the TSwitch™ cDNA instead of mRNA or total RNA can also provide information similar to standard Northern blot.
The TSwitch™ cDNA is very suitable for rapid amplification of cDNA ends (RACE). The kit can also be used for the synthesis of the 1st strand cDNA with adaptor primers at the 5' and 3' ends from mRNA or total RNA.