Genome-wide analysis of APA based on 3t-seq

A genome-wide, wide-ranging analysis technology, applied in the field of RNA PAS detection, which can solve the problems of inability to completely eliminate polyA, large RNA damage and loss, and complicated RNA level operations.

Inactive Publication Date: 2015-08-12
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, the SAPAS method cannot completely eliminate the impact of unexcision of polyA on sequencing. The RNA level operation in the 3P-Seq method is complicated. After multi-step RNA ligation and enzyme digestion, the damage and loss of RNA are also relatively large, which is not suitable for Small sample library construction
Moreover, this method is difficult and expensive
In addition, there are limitations in the fragmentation method during the operation, that is, the method of RNase T1 enzyme fragmentation in the 3P-Seq method and the direct heating fragmentation of RNA in the SAPAS method cannot achieve random fragmentation without damaging the RNA.

Method used

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  • Genome-wide analysis of APA based on 3t-seq
  • Genome-wide analysis of APA based on 3t-seq
  • Genome-wide analysis of APA based on 3t-seq

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Experimental program
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Embodiment Construction

[0024] The buffer composition used in the specific embodiment is as follows:

[0025] Reagent storage mother solution required for preparing buffer solution (can be purchased from a commercial company, or purchase related reagents to prepare by yourself):

[0026]

[0027] Binding buffer 1 (20ml):

[0028] DEPC treated water 10ml

[0029] 5M NaCl 8ml

[0030] 0.5M EDTA (PH 8.0) 2ml

[0031] Binding buffer 2 (20ml):

[0032]

[0033]

[0034] Wash buffer A (20ml):

[0035]

[0036] Wash buffer B (20ml):

[0037]

[0038] Wash buffer C (20ml):

[0039]

[0040] Wash buffer D (20ml):

[0041]

[0042] The mother solutions for the following washing buffers are all derived from the products of relevant commercial companies, and the mother solutions need to be purchased from the relevant companies to prepare according to the following formula. It is recommended to prepare freshly for each experiment:

[0043] Wash Buffer 1 (400 μl):

[0044] DEPC treate...

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Abstract

The invention relates to a method for detecting a PAS (Poly A Site) of an RNA (Ribose Nucleic Acid), and provides a method for analyzing APA (Alternative Polyadenylation) based on 3T-seq (TT-mRNA Terminal Sequencing) within a whole-genome range. The method for analyzing APA comprises the following steps: preparing magnetic beads containing oligo dT, i.e., mixing an oligo dT primer modified by 5' end biotin and the magnetic beads with streptavidin; mixing the magnetic beads with total RNA and screening RNA containing Poly A; performing reverse transcription, synthesizing the second strand of a double-stranded cDNA and breaking the double-stranded cDNA; removing the Poly A structure; after terminal modification, connecting a sequencing linker; and recovering libraries. According to the method for analyzing APA, RNA horizontal operation is reduced, a Poly A sequence is horizontally removed from the DNA, and the influence of the Poly structure on sequencing is eliminated; double-stranded DNA breaking enzyme is selected for treatment, and on the premise that the quality of the libraries is not affected, the DNA is broken horizontally; the experiment process is simplified, the experiment difficulty and cost are lowered and the method for analyzing APA is wide in application range.

Description

technical field [0001] The present invention relates to a method for detecting RNA PAS (Poly A Site), in particular to a method for analyzing APA (Alternative Polyadenylation) in the whole genome based on 3T-seq (TT-mRNA Terminal Sequencing) technology. Background technique [0002] In the human genome, more than half of the genes have multiple polyadenylation sites (PAS, Poly A Site). During pre-mRNA maturation, maturation with a 3' non-transcoding region (3' UTR) of varying lengths is generated from a coding region by altering the cleavage site and polyadenylation site (PAS) of the pre-mRNA mRNA, this situation is called Alternative Polyadenylation (APA, Alternative Polyadenylation). APA plays a vital role in the regulation of cell proliferation, differentiation, transport and development, as well as the occurrence of diseases such as tumor development and other physiological and pathological processes. [0003] There are many existing techniques for studying APA changes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2563/143C12Q2525/173C12Q2525/131
Inventor 赵小东邵志峰康亚妮赖登攀吴俊
Owner SHANGHAI JIAOTONG UNIV
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