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RNA library construction method

A construction method and library technology, applied in the field of RNA sequencing library construction, can solve problems such as cumbersome library construction process, and achieve the effect of simple operation and saving man-hours

Active Publication Date: 2021-01-05
天津诺禾致源生物信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The main purpose of the present invention is to provide a method for constructing an RNA (initial amount of 100ng-1000ng) library, to solve the cumbersome problem of the library construction process in the prior art

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. One-step method to complete RNA fragmentation and reverse transcription reaction (Novizan kit, product number: NR604)

[0049] (1) Use 19.5 μL fragmentation buffer (Fragment buffer) to elute the enriched RNA sample, heat at 94°C for 8 minutes to complete the RNA fragmentation reaction, and perform reverse transcription reaction immediately;

[0050] (2) Prepare the first-strand cDNA synthesis reaction system according to the following table:

[0051] Table 1:

[0052]

[0053] (3) Carry out the first-strand cDNA synthesis reaction in a PCR instrument:

[0054] Table 2:

[0055]

[0056] (4) Immediately purify with 1.8X DNA clean beads (Clean beads), then use 16μL ddH 2 O to elute the beads.

[0057] 2 single chain header connections

[0058] (1) configure the reaction solution by the following components:

[0059] table 3:

[0060]

[0061] Incubate at 60°C for 1 hour, and incubate at 80°C for 10 minutes to denature.

[0062] (2) Use magnetic beads t...

Embodiment 2

[0082] Compared with the common RNA-seq library construction method (that is, the method of building a library by synthesizing double-stranded cDNA mentioned in the background technology), using the method of the present invention in the test of tobacco samples, the quality inspection results of the libraries constructed by the two methods were respectively Such as Figure 3A with Figure 3B shown.

[0083] The analysis results of sequencing data are shown in Table 7. Q20 and Q30 are above 97% and 92% respectively, the mapping rate is above 94%, and other indicators meet the requirements. Figure 4 Shown are the difference graphs of gene expression detected after the libraries are constructed by the two methods, and it can be seen from the graphs that the results of the method of the present invention are consistent with those of the traditional method.

[0084] Table 7:

[0085]

Embodiment 3

[0087] Compared with the common RNA-seq library construction method, using the method of the present invention in the test of human samples, the quality inspection results of the libraries constructed by the two methods are as follows: Figure 5A with Figure 5B shown.

[0088] The analysis results of sequencing data are shown in Table 8. Q20 and Q30 are above 96% and 91% respectively, the mapping rate is above 95%, and other indicators meet the requirements. Image 6 Shown are the difference graphs of gene expression detected after the libraries are constructed by the two methods, and it can be seen from the graphs that the results of the method of the present invention are consistent with those of the traditional method.

[0089] Table 8:

[0090]

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Abstract

The invention provides a RNA library construction method. The construction method comprises the following steps: carrying out reverse transcription on fragmented RNA by adopting a reverse transcription primer to obtain a first strand cDNA, wherein the reverse transcription primer sequentially comprises a known sequence and a random sequence from 5' end to 3' end, carrying out single-chain linker connection on the first chain cDNA by adopting cyclase to obtain a connection product, carrying out PCR amplification on the connection product to obtain an RNA library, introducing a first sequencingprimer sequence while RNA is subjected to reverse transcription to synthesize first-chain cDNA, then directly carrying out single-chain linker connection through cyclase to introduce a second sequencing primer sequence, and finally, designing a proper amplification primer according to the first sequencing primer sequence and the second sequencing primer sequence to carry out PCR amplification on alinker connection product so that RNA libraries can be obtained. According to the method, synthesis of second chain cDNA is not needed, chain specificity is directly achieved without other digestionprocesses, operation is easy, and working hours are saved.

Description

technical field [0001] The invention relates to the field of RNA sequencing library construction, in particular to a method for constructing an RNA library. Background technique [0002] Transcriptome sequencing technology, RNA-seq is an important tool for studying biological gene expression, and this technology is widely used in the analysis of gene expression profiles of different species. The RNA-seq library construction kits that have emerged in recent years mainly focus on the research of RNA strand specificity. The library construction process needs to extract total RNA and enrich mRNA, synthesize the first strand cDNA by reverse transcription, and use dUTP to synthesize the second strand cDNA. Subsequently, the end of the double-stranded cDNA was blunted, adenine A base was added, and then the adapter was ligated, and the cDNA containing uracil U base was digested and then amplified by PCR to complete the library construction. [0003] The process of building a datab...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C40B40/08
CPCC40B50/06C40B40/06C40B40/08
Inventor 李新马玉李瑞强赵桂仿
Owner 天津诺禾致源生物信息科技有限公司
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