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Transcriptome library and construction method thereof

A construction method and library construction technology, applied in the field of high-throughput sequencing library construction, can solve the problems such as difficult to realize transcriptome library construction of trace starting cells

Inactive Publication Date: 2015-10-28
BEIJING NOVOGENE TECH CO LTD
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Problems solved by technology

[0009] The main purpose of the present invention is to provide a transcriptome library and its construction method to solve the problem in the prior art that it is difficult to realize the construction of transcriptome library of trace starting cells

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  • Transcriptome library and construction method thereof
  • Transcriptome library and construction method thereof

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Embodiment Construction

[0026] It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with examples.

[0027] As mentioned in the background technology section, the current method of constructing a transcriptome library is to directly use the ribosomal RNA removed from the total RNA (to avoid the impact of a large amount of ribosomal RNA on the sequencing), and the isolated mRNA is obtained by reverse transcription. cDNA is used for library construction without the step of PCR amplification in the middle, which reduces the bias or error rate caused by PCR amplification, so that the constructed transcriptome library can relatively truly reflect the transcription of cells or tissues in a certain state situation. However, the above-mentioned construction method has higher requirements on the amount of total RNA in the sample...

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Abstract

The invention provides a transcriptome library and a construction method thereof. The construction method comprises the following steps: total RNA of a sample to be detected is adopted as a template; reverse transcription is carried out with MMLV reverse transcriptase, such that double-stranded cDNA of the mRNA in the total RNA is obtained; the double-stranded cDNA is amplified, such that amplified cDNA is obtained; and the amplified cDNA is used in fragmented library construction, such that the transcriptome library is obtained. According to the invention, mRNA in the total RNA is reverse-transcribed into full-length fragment double-stranded cDNA with the MMLV reverse transcriptase, and the double-stranded cDNA is amplified, such that a DNA amount needed by library construction is reached, and subsequent segmented library construction can be realized smoothly. With the method, a complete full-length transcript can be enriched; cDNA can be amplified with high sensitivity and no preference; and the fullness of the transcripts of original mRNA can be maintained, such that the construction of trace sample transcriptome library can be realized.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing library construction, in particular to a transcriptome library and a construction method thereof. Background technique [0002] Transcriptome sequencing (Transcriptome sequencing) mainly uses high-throughput sequencing to study the mRNA transcribed from a specific tissue or cell during a certain period. Transcriptome sequencing can comprehensively and quickly obtain almost all transcripts and gene sequences of a specific cell or tissue of a species in a certain state, which can be used to study gene structure and gene function, alternative splicing and prediction of new transcripts, etc. [0003] At present, in the step of constructing the transcriptome library, ribosomal RNA is usually removed from the total RNA first to avoid the impact of a large amount of ribosomal RNA on the sequencing. The ribosome-removed RNA is reverse-transcribed using random primers to obtain cDNA. This not on...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06
Inventor 王大伟李宗文蒋智朱海浩李富威刘运超李明洲
Owner BEIJING NOVOGENE TECH CO LTD
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