mRNA fragmentation method and method for constructing sequencing library based on same
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A sequencing library and fragmentation technology, which is applied in the field of molecular biology, can solve problems such as many adapter connection steps, cumbersome library construction process and large differences in library construction costs, and long overall library construction time
Active Publication Date: 2016-10-05
MGI TECH CO LTD
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Abstract
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Problems solved by technology
The method of adding joints is different, the cumbersomeness of the database construction process and the cost of database construction will also vary greatly
[0004] In order to solve the problems of too many adapter ligation steps and long overall library construction time in the existing RNA sequencing library construction, the present invention is proposed.
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Embodiment 1
[0088] Example 1 Construction of RNA sequencing library without PCR amplification (PCR free)
[0089] 1. Purified mRNA
[0090] (1) DNA probe hybridizes to rRNA in total RNA
[0093] (2) RNaseH digestion of rRNA hybridized with DNA probe
[0094] product of previous step
5μl
10xRNase H buffer
1μl
RNase H (5U / μl)
2μl
RF water
2μl
total capacity
10μl
[0095] Reaction conditions: 37°C for 30min
[0096] (3) DNaseI digestion of DNA probes
[0097] product of previous step
10μL
10xDNaseI buffer
2μL
DNaseI (2U / μl)
5μL
[0098] RF water
3μL
total capacity
20μL
[0099...
Embodiment 2
[0158] Example 2 Construction of RNA sequencing library requiring PCR amplification
[0159] 1. Purification of mRNA
[0160] (1) Hybridization between DNA probe and total RNA
[0161] total RNA
2μg
Probe MIX (100uM, i.e. oligonucleotide probe)
1μl
5x Hybridization Buffer
1μl
Add RF water to total volume
5μl
[0162] Reaction conditions: 95°C for 2 min, 0.1°C / sec (gradient cooling), 22°C for 3 min.
[0163] (2) RNaseH digestion of rRNA hybridized with DNA probe
[0164] product of previous step
5μl
10xRNase H buffer
1μl
RNase H (5U / μl)
2μl
RF water
2μl
total capacity
10μl
[0165] Reaction conditions: 37°C for 30min
[0166] (3) DNaseI digestion of DNA probes
[0167] product of previous step
10μl
10xDNaseI buffer
2μl
DNaseI (2U / μl)
5μl
RF water
6μl
total capacity
20μl
[0168] Reaction cond...
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Abstract
The invention relates to an mRNA fragmentation method and a method for constructing a sequencing library based on the mRNA fragmentation method. The mRNA fragmentation method provided by the invention employs forward and reverse bridge-type probes, realizes breakage of an mRNA sample through one-step reverse transcription and connection reaction, and introduces two terminal linkers during reverse transcription so as to obtain a cDNA library with linkers at two terminals. The cDNA library with the linkers at the two terminals can directly undergo cyclization reaction or undergo PCR amplification before cyclization reaction, so a sequencing library for single-stranded cyclic nucleic acids can be obtained; or a sequencing library for single-stranded nucleic acids can be obtained by directly subjecting the cDNA library to amplification. When the mRNA fragmentation method is applied to construction of the sequencing library, tedious steps like restoration of a tail terminal and arrangement of a linker on one end at first and another linker on the other end next in traditional construction of libraries can be omitted; experimental flow is greatly simplified; a library construction period is shortened; and library construction cost is greatly reduced.
Description
technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for fragmenting an mRNA sample, a method for constructing a sequencing library based on the mRNA fragmentation method, and the application of the constructed sequencing library in high-throughput sequencing. Background technique [0002] Next-generation sequencing technology, also known as high-throughput sequencing technology, is characterized by high sequencing throughput, short sequencing time and low sequencing cost. RNA-seq, also known as transcriptome sequencing technology, uses high-throughput sequencing technology to detect the sequence of mRNA, small RNA, noncoding RNA, etc., or some of them, to detect their expression. Level. [0003] Usually, when constructing an RNA sequencing library, it is necessary to purify mRNA first, then fragment the purified RNA sample, and finally add sequencing adapters to both ends of the sequence fragment to be tested. ...
Claims
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