Method for researching APA within strand-specific whole-genome range

Abstract

The invention discloses a method SS-3T-seq for researching APA within a strand-specific whole-genome range. The method is used for researching APA changes within a whole-genome range and comprises the following steps: firstly building a special magnetic bead containing oligo dT; screening RNA containing a polymer A by using the magnetic bead; synthesizing a first-stranded cDNA by reverse transcription on the magnetic bead, and replacing normal dCTP with methylated dCTP in a reverse transcription system; synthesizing double-stranded cDNA by using special dNTP (dTTP is replaced by dUTP); after breaking, taking the remaining part on the magnetic bead as a fragment containing an APA locus; removing the polymer A structure by Gsu I enzyme excision; releasing a target fragment from the magnetic bead; modifying the two ends and adding an Illumina sequencing joint; screening fragments according to size and performing USER enzyme treatment to remove the dUTP in the second strand; leaving the first strand for PCR amplification to obtain a library which contains strand information and can be used for Illumina next-generation sequencing. The method is simple in experiment operation, has the advantages of reducing loss caused by complex steps and also reducing the experiment difficulty and the manufacturing cost, and is quite suitable for popularization.

Description

technical field [0001] SS-3T-seq (Strand-specific 3'Terminal Sequencing) of the present invention relates to a method for constructing a high-throughput sequencing library of the 3' terminal non-coding region of strand-specific mRNA, which is used to study selective multimerization on a genome-wide scale Alternative Polyadenylation (APA). Background technique [0002] There are many existing technologies for studying APA changes on a genome-wide scale, but the methods and principles are similar, and the two most representative ones are listed here. [0003] figure 1 with figure 2 Experimental schematics showing both approaches: [0004] figure 1 It is SAPAS (Sequencing APA Sites). After the RNA is directly heated and cleaved, the first-strand cDNA is reverse-transcribed with a primer containing oligo dT, and the double-stranded cDNA is synthesized by a strand displacement method. Then, the mutated oligo dT (TTTTCTTTTTTCTTTTTT) was connected to the sequencing adapter as...

AI Technical Summary

Problems solved by technology

The disadvantage of the 3P-Seq method is that the RNA level operation is complicated, and it needs to go through multi-step RNA connection and enzyme digestion, and the damage and loss of RNA are also large. It is not suitable for the construction of a small number of sample libraries, and this method is difficult and expensive. higher

[0006] In addition, the fragmentation methods of the above two methods have limitations. The methods of RNase T1 enzyme fragmentation (3P-Seq) and RNA direct heating fragmentation (SAPAS) cannot achieve the simultaneous fragmentation without damaging the RNA.

Moreover, many genomes have both positive and negative strand transcripts in the same region of the genome, and the prior art methods do not know the orientation information of the transcript strands, that is, the sequence obtained by sequencing is from the positive strand or the negative strand of the DNA double strand

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