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Construction method and application of sequencing library of circular small non-coding RNA (Ribonucleic Acid)

A sequencing library and construction method technology, applied in the field of molecular biology, to achieve the effect of high data accuracy, high quality, and good reliability

Pending Publication Date: 2021-11-23
深圳市龙华区中心医院
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the high-throughput sequencing platform based on cPAL does not have a corresponding construction method for circular small non-coding RNA library. A method for high-throughput sequencing libraries, and techniques to enable such libraries to be sequenced by the cPAL method

Method used

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Embodiment Construction

[0026] The principles and features of the present invention are described below, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

[0027] Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer.

[0028] 1. Materials

[0029] Human embryonic kidney epithelial cells HEK293 were purchased from ATCC (American Type Culture Collection) in the United States and preserved in this experiment.

[0030] 2. Main reagents

[0031] Cell culture medium DMEM, fetal bovine serum (FBS), and deoxyribonuclease (DNase I) were purchased...

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Abstract

The invention relates to a construction method and application of a sequencing library of circular small non-coding RNA (Ribonucleic Acid). The construction method comprises the following steps: (1) culturing cells; (2) extracting total RNA (Ribonucleic Acid) from the cells; (3) extracting RNA by using a small RNA extraction kit, and removing 28S and 18S rRNA; (4) further removing 5S and 5.8 S rRNA by an RAP method; (5) removing linear RNA; and (6) constructing the sequencing library of the circular small non-coding RNA. The constructed sequencing library of the circular small non-coding RNA has the advantages of few rRNA residues, high detection efficiency of the circular RNA, high sequencing flux, better data repeatability and high success rate of verification of a sequencing result. The whole method is simple and convenient to operate, low in cost and time consumption, high in stability and very good in repeatability and reliability.

Description

technical field [0001] The invention relates to a method for constructing a circular small non-coding RNA sequencing library and its application, and belongs to the technical field of molecular biology. Background technique [0002] In the human genome, about 70% of the sequences can be transcribed into RNA, but most of them cannot be translated into proteins. These noncoding RNAs (noncoding RNA, ncRNA) are divided into long noncoding RNA (>200nt, long noncodingRNA, lncRNA) and Small non-coding RNA (<200nt, small non-coding RNA, sncRNA). [0003] Circular RNA (ciucular RNA, circRNA) is a new member of the RNA family that is different from traditional linear RNAs. Coding RNA molecules. [0004] The high-throughput sequencing platform based on combinatorial probe-anchor ligation (cPAL) is a new type of high-throughput sequencing platform, and the library required for sequencing on this platform is a single Chain ring molecules, through the rolling circle replication o...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C40B50/06
CPCC12Q1/6806C40B50/06C12Q2527/125C12Q2523/308C12Q2521/327C12Q2521/345C12Q2535/122
Inventor 周仁龙肖锡林张勇刚刘陈贺李琼
Owner 深圳市龙华区中心医院
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