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High-throughput transcriptome library construction method

A library construction and transcriptome technology, applied in chemical libraries, combinatorial chemistry, recombinant DNA technology, etc., can solve the problems of high price, increase the time and cost of library construction, etc.

Inactive Publication Date: 2018-09-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

Among them, expensive nucleic acid purification magnetic beads are generally used in the process of mRNA purification and subsequent nucleic acid purification, and special nucleic acid fragmentation kits are generally used in mRNA fragmentation, and qPCR is used to test samples before sequencing on the machine. Absolute quantification done, these steps significantly increase the time and cost of library construction

Method used

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Examples

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Embodiment 1

[0069] Example 1: Transcriptome library construction of Miscanth total RNA

[0070] Specific steps are as follows:

[0071] (1) mRNA purification

[0072] 1. Dilute 15 μg of total RNA with Nuclease-free H 2 O to dilute to 50 μL.

[0073] 2. Heat the RNA sample at 65°C for 2 minutes, then place it on ice immediately. (destruction of secondary structure)

[0074] 3. Put 25μL Olgio(dT)beads in 120μL Binding Buffer, and pipette up and down with the pipette tip to clean. After washing twice, place the washed Olgio(dT)beads in 50μL Binding Buffer. (If multiple samples are processed at the same time, this step can be performed at the same time. For example, wash 200ul beads at the same time for 8 samples.)

[0075] 4. Mix the above 50 μL Olgio(dT) beads with 50 μL total RNA, and incubate by vortexing at room temperature for 10 min.

[0076] 5. Put the above-mentioned centrifuge tube on the magnetic stand and let it stand, and remove the supernatant after the supernatant is cla...

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Abstract

The invention relates to the molecular biology and biotechnology field, in particular to a high-throughput transcriptome library construction method. The method utilizes reverse transcription cDNA first strand synthesized Buffer as a fragmentation reaction system, so that fragmentation reaction and reverse transcription first strand synthesis can be linked up seamlessly, and the experimental operation steps can be simplified. During purification and recovery of the cDNA second strand synthesized and end repaired product in library construction, nucleic acid is utilized to purify magnetic beadsrepeatedly. During equivalent pooling of sequencing samples, precise pooling of samples can be achieved directly according to the Qubit detection result without absolute quantification of the samplesby qPCR. The above-mentioned improvements can effectively reduce the workload in the library construction process, and at the same time also greatly lowers the library construction cost.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a method for constructing a high-throughput transcriptome library. Background technique [0002] Transcriptome refers to the collection of all transcripts of a specific cell under a certain functional state or physiological condition. In a broad sense, it refers to the sum of all RNAs that can be transcribed; in a narrow sense, it refers to the collection of all mRNAs. [0003] Since protein is the main bearer of cellular functions, proteome is the most direct description of cell function and state, but there are certain limitations in current protein experimental techniques, which makes transcriptome the main means of studying gene expression. Transcriptome is an inevitable link between genomic information and proteome that connects biological functions. It is the basis and starting point for the study of gene function and structure. It has been widely used in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B50/06
CPCC12N15/1013C40B50/06
Inventor 陈翠霞王伟栋刘文雪葛春霞
Owner SHANDONG AGRICULTURAL UNIVERSITY
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