CirclSeq – Automatable and relibale preparation of a DNA library in a one-vial reaction for unbiased quantification of mRNA

The analysis of the mRNA content of a cell or a tissue via sequencing provides a method for functional analysis. In common protocols, prior to the sequencing procedure itself the mRNA has to be reverse transcribed into cDNA, followed by random shearing into cDNA fragments, linker ligation, and amplification via PCR. The library of PCR amplicons can then be sequenced by various methods of next generation sequencing (NGS). In many protocols, the primers used for the reverse transcription (RT) or ligation have to be removed before sequencing. Typically, this is achieved by performing a polyacrylamide gel electrophoresis, which suffers from poor quantitative yield and poor discrimination between molecules of similar size. Additionally, PCR amplification can lead to biased quantification of rare mRNA species. The present invention allows overcoming these problems by using a new protocol, which consists of the following steps:
1) RT of mRNA into cDNA in presence of dUTP
2) RNA digestion and 3’ end blocking of RT primer with ddTTP
3) Enzymatic cleavage at positions of dUTP incorporation
4) cDNA circularization
5) NGS

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