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Method for building transcriptome sequencing library, corresponding joint sequence and kit

A transcriptome sequencing and linker sequence technology, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve the problems of high connection efficiency, low double-link connection efficiency, unstable concentration and ratio, etc., to achieve high efficiency The effect of connecting, not easy to connect self-connection

Inactive Publication Date: 2018-11-06
GUANGZHOU MICROCHIP BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The main advantages and disadvantages of these three kinds of joints are that the efficiency of double-linked joints with flat ends is moderate, and it is easy to form joint self-connection; the connection efficiency of double-linked joints with Y-shaped structure is low, and an additional annealing step is required for the manufacturing process, and two steps before annealing The concentration and ratio of single strands are often unbalanced and unstable due to quantitative problems; the U-shaped structure of the single-chain linker is a single-chain structure, which naturally forms a partially double-stranded stem-loop structure without additional annealing. High efficiency, not easy to generate adapter self-ligation, but need to use enzymes to open the U-shaped structure before PCR amplification of the library, increasing the experimental steps and reagent costs

Method used

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  • Method for building transcriptome sequencing library, corresponding joint sequence and kit
  • Method for building transcriptome sequencing library, corresponding joint sequence and kit
  • Method for building transcriptome sequencing library, corresponding joint sequence and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The length of embodiment 1 carbon chain modification

[0035] 1. Cell culture

[0036]Use GIBCO's DMEM and 10% FBS serum to culture 293T cells at 37°C and 5% CO2. After 2 days of normal culture, the cell density is about 60-80% (40,000-50,000).

[0037] 2. Total RNA Extraction

[0038] Total RNA was extracted according to Qiagen's QIAamp RNA Blood Mini Kit instructions.

[0039] 3. Transcriptome sequencing library construction

[0040] 100ng initial amount (total RNA of cells), using NEBNext RNA Ultra-fast Library Preparation Kit-Illumina for library construction, the main steps are: RNA fragmentation (the average fragment length after fragmentation is 200bp); one-strand reverse transcription; Two-strand reverse transcription; double-strand cDNA end repair and purification; 3' end addition and purification; sequencing adapter ligation and purification; PCR amplification and purification, and a transcriptome sequencing library was obtained. The specific steps were ca...

Embodiment 2

[0050] Example 2 The number of units modified by the carbon chain

[0051] 1. Cell culture

[0052] Concrete method is with embodiment 1.

[0053] 2. Total RNA Extraction

[0054] Concrete method is with embodiment 1.

[0055] 3. Transcriptome sequencing library construction

[0056] Concrete method is with embodiment 1.

[0057] The specific steps were carried out according to the instructions of the ultra-rapid transcriptome library preparation kit. The sequence adapter sequences of different numbers of long carbon chain modification units are shown in Table 4, and the concentration of the storage solution for the ligation reaction was 15 μM.

[0058] Table 4

[0059]

[0060] 4. Transcriptome sequencing library quality inspection

[0061] Concrete method is with embodiment 1.

[0062] 5. Result analysis:

[0063] The quality inspection results of the transcriptome sequencing library showed that using NEB adapters as positive controls, 1 C18 (18 consecutive long c...

Embodiment 3

[0067] Embodiment 3 linker concentration

[0068] 1. Cell culture

[0069] Concrete method is with embodiment 1.

[0070] 2. Total RNA Extraction

[0071] Concrete method is with embodiment 1.

[0072] 3. Transcriptome sequencing library construction

[0073] Concrete method is with embodiment 1.

[0074] The specific steps were carried out according to the instructions of the ultra-rapid transcriptome library preparation kit. The sequences of the sequencing adapters are shown in Table 6. The concentrations of the stock solution for the ligation reaction were 5 μM, 10 μM, 15 μM and 20 μM, respectively.

[0075] Table 6

[0076]

[0077] 4. Transcriptome sequencing library quality inspection

[0078] Concrete method is with embodiment 1.

[0079] 5. Result analysis

[0080] The results of the quality inspection of the transcriptome sequencing library showed that, using the NEB adapter as a positive control, using a C18 (18 continuous long carbon chain modification) s...

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Abstract

The invention discloses a method for building a transcriptome sequencing library, a corresponding joint sequence and a kit. The joint sequence used for building the transcriptome sequencing library ingeniously uses the chemical modification of C3-C21 long carbon chains; the chemical modification is applied to a stem loop structure sequencing joint formed by single chains. Compared with the existing mainstream library building joint, the joint has the advantages that the joint dimerization cannot be easily generated; the additional double-chain annealing is not needed; the use of enzymes for opening loop structures in the stem loops is not needed; on the basis of not influencing the later stage PCR amplification, the reaction time is greatly saved, and the reagent cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a transcriptome sequencing library, a corresponding linker sequence and a kit. Background technique [0002] With the development of modern science and technology, the research of life science has entered the era of omics. Gene and genome sequencing technology has become an indispensable means in modern life science research, especially in genomics research. In recent years, the rapid development of next-generation genome sequencing technology has brought about unprecedented prosperity in genomics research. Next-generation sequencing technology has been widely used in various fields of life sciences, agronomy, medicine, environmental protection, forensic science and other fields. With the rapid development of next-generation high-throughput sequencing technology, transcriptome sequencing has become an important means to study gene expression an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C40B50/06C12Q1/6869C12N15/11C12N15/10
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2521/107C12Q2525/191C12Q2531/113
Inventor 曹亮束文圣吴胜标王璋郝祎琪
Owner GUANGZHOU MICROCHIP BIOTECH CO LTD
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