Balance joint with molecular barcode and method for quickly constructing transcriptome library
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A molecular barcode and transcriptome technology, applied in the field of data analysis, reagents and kits for balancing adapters and/or internal reference standard RNA, can solve the problem of indistinguishable duplication, pollution, etc., to solve the problem of data waste and accurate quantification. Effect
Active Publication Date: 2020-10-23
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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[0006] In view of this, the present invention is committed to providing a balanced adapter with a molecular barcode, which can completely solve the problem that the prior art cannot distinguish between the natural repetition of the sample itself and the duplication generated by the PCR process, and the problem of primer dimers, Contamination issues introduced by non-specific annealing fragments, enabling accurate quantification of transcripts
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Embodiment 1
[0104] Example 1 prepares internal reference standard RNA
[0105] (1) Using unmethylated lambda DNA (GenBank-EMBL Accession Number: J02459) as a template, design 9 pairs of primers.
[0106] The primer sequences used in the following examples are shown in Table 2, F represents the PCR forward primer, and R represents the PCR reverse primer. The underlined part of the 5' end sequence of the forward primer is the T7 promoter sequence and the 3 Gs added after the promoter sequence. The underlined part of the 5' end sequence of the reverse primer is 20 polyTs.
[0107] Table 2
[0108]
[0109] (2) PCR amplification obtained 5 products with a size of 1840bp and 4 products with a size of 640bp. Among them, the GC contents of 5 1840bp products were: 49% for P1840-1, 48% for P1840-2, 49% for P1840-3, 57% for P1840-4, 58% for P1840-5, and 4 640bp products The GC contents of the samples were: 52% for P640-1, 41% for P640-2, 59% for P640-3, and 56% for P640-4.
[0110] (3) Usin...
Embodiment 2
[0112] Example 2 Transcriptome library construction
[0113] 1. mRNA fragmentation
[0114] (1) Take 0.1 μg of total RNA extracted from the mouse C2C12 cell line, add the internal reference standard RNA prepared in Example 1 (the amount added is 1% of the total amount of mRNA, and the amount of mRNA is calculated according to 2% of the total amount of RNA) ),use mRNACapture Beads captures mRNA and obtains 10ul of samples.
[0115] (2) Prepare the fragmentation reaction system in a 0.2ml PCR tube with the mRNA obtained in the previous step as shown in Table 3:
[0116] table 3
[0117]
[0118] Then put the PCR tube into the PCR instrument, incubate at 85°C for 6min, and store at 4°C.
[0119] 2. Synthesis of cDNA by reverse transcription and addition of linkers
[0120] (1) Add the following ingredients in Table 4 to the above 17 μL mRNA fragment sample:
[0121] Table 4
[0122]
[0123] The BBAs in Table 4 are mixed in the same molar ratio by two types of BBA-L...
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Abstract
The invention provides a balance joint with a molecular barcode and a method for quickly constructing a transcriptome library. The balance joint with the molecular barcode is formed by mixing two kinds of balance joints with different lengths, and the balance joint sequentially comprises a sequencing library PCR primer recognition sequence, a molecular bar code sequence and a terminal base sequence from the 5' terminal to the 3' terminal; and the long balance joint also includes an intermediate base sequence. The joint can thoroughly solve the problem of data pollution caused by duplication, non-specific annealing and other reasons generated in the PCR process, and overcomes the problems of transcriptome library construction by using SMART technology product lines and base imbalance duringLane sequencing. The invention also provides a method for quickly constructing the transcriptome library by using the joint and adding an internal reference standard substance, so that the steps aresimplified, and the library construction time is shortened to a great extent; and the internal reference standard substance is added, so that the deviation caused by a data result can be subjected tostandardized correction, and the reliability of subsequent analysis is ensured.
Description
technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a balanced linker with a molecular barcode and its application, a method for quickly constructing a transcriptome library by using a balanced linker and an internal reference standard RNA, including a balanced linker and / or an internal reference standard RNA Reagents and kits, and data analysis methods. Background technique [0002] With the development and progress of science and technology, life science research has entered the post-genome era. As a very important omics research in the post-genome era, transcriptome research provides important means and methods for the study of gene expression and transcriptional regulation, and is also an important way to discover functional genes. The concept of transcriptome was first proposed by Velcuescu et al. in 1995 when studying yeast gene expression. Transcriptome in a broad sense refers to the sum of all RNAs tr...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C40B50/06C12Q1/6869
CPCC12N15/1093C12Q1/6869C40B50/06C12Q2525/191
Inventor 崔鹏秦锐林强范伟
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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