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Method for generating a RNA-sequencing library

A technology for sequencing, DNA strands, applied in the field of preparing strand-specific RNA sequencing libraries, which can solve problems such as time-consuming, UNG reaction is not 100% effective, and misinterpretation of RNA sequencing data.

Inactive Publication Date: 2018-03-27
QIAGEN GMBH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The more widely used method of using dUTP in addition to dNTPs in second-strand DNA synthesis requires an additional UNG digestion step after adapter ligation, making the library construction process more complex and time-consuming
Also, like any enzymatic reaction, the UNG reaction is not 100% efficient
Residual second-strand cDNA may remain even after UNG digestion, which can lead to misinterpretation of RNA-Seq data

Method used

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  • Method for generating a RNA-sequencing library
  • Method for generating a RNA-sequencing library
  • Method for generating a RNA-sequencing library

Examples

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Embodiment

[0191]RNA from HeLa cells was extracted with RNeasy kit (QIAGEN), and PolyA+mRNA was enriched with GeneRead Pure mRNA Kit (QIAGEN). 86ng of PolyA+ mRNA was then used in each RNA-Seq library preparation reaction according to the following protocol:

[0192] 32 μl of mRNA (total amount: 86 ng) was mixed with 8 μl of qScript Flex Reaction Mix (5X) (QuantaBiosciences), 2 μl of random 8mer oligonucleotides (200 μM, IDT). The random oligonucleotides were natural oligonucleotides ("Control") or oligonucleotides with a C3 spacer at the 5' ('Mod', / 5SpC3 / NNN NNN NN, IDT)), which would block Ligation of the resulting cDNA to the sequencing adapters.

[0193] The mRNA / random oligonucleotide mixture was heated at 94°C for 15 minutes to fragment the RNA to an average size of approximately 100-200 bp. After heat-mediated fragmentation, the mixture was cooled on ice.

[0194] Subsequently, reverse transcription (RT) components were added: 2 μl RNAse inhibitor (4 U / μl, QIAGEN), 2 μl dNTPs ...

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Abstract

The invention refers to a novel method of preparing strand-specific RNA-sequencing libraries that can be used to identify DNA coding and non-coding strands that are transcribed to RNA. Such strand-specific RNA-sequencing libraries are especially useful in discovering anti-sense RNA and non-coding RNA. Random primer oligonucleotides, covalently coupled to a moiety, which blocks ligation,are used for RT reaction or the subsequent generation of the second DNA strand so that only one strand of the generated double-stranded DNA is ligated to sequencing adapters at the 5 nucleotide and sequenced by paired-end sequencing.

Description

technical field [0001] The present invention relates to a novel method for preparing strand-specific RNA sequencing libraries that can be used to identify coding and noncoding strands of DNA transcribed into RNA. This strand-specific RNA sequencing library is especially useful for discovering antisense RNA and non-coding RNA. Random primer oligonucleotides covalently coupled to the part of the blocking ligation are used in the RT reaction or subsequent generation of the second DNA strand such that only one strand of the resulting double-stranded DNA is at the 5' nucleotide Ligated to sequencing adapters and sequenced by paired-end sequencing. Background technique [0002] In addition to the mRNAs covering 1.5% of higher eukaryotic genomes, a number of noncoding RNAs with widely varying expression levels have been identified. The biological functions of these novel transcripts are largely unknown and represent a new area of ​​research that requires high-throughput transcrip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6869
CPCC12Q1/6869C12Q2521/107C12Q2521/131C12Q2521/501C12Q2525/186C12Q2525/191C12Q1/6855
Inventor 方南伯恩哈德·诺尔卡特娅·海茨
Owner QIAGEN GMBH
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