Construction method and application of sequencing library of long-chain non-coding RNA

A long-chain non-coding, sequencing library technology, applied in the fields of biology and gene sequencing

Pending Publication Date: 2020-07-07
ZHEJIANG ANNOROAD BIO TECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this approach inevitably excludes information from other RNAs without poly(A) tails

Method used

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  • Construction method and application of sequencing library of long-chain non-coding RNA
  • Construction method and application of sequencing library of long-chain non-coding RNA
  • Construction method and application of sequencing library of long-chain non-coding RNA

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Embodiment 1

[0059] The single-cell lncRNA transcriptome sequencing libraries of two human samples (numbering CS20161012 and CS20161013), one rat and one mouse were respectively constructed by using the method of the embodiment of the present invention, as follows:

[0060] 1. RNase H method to remove rRNA

[0061] 1.1 Collect single cells from each sample separately, put 0.5 μl single cells in a 0.2ml thin-walled PCR tube, which contains 0.1 μl rRNAProbe (H (human) / M (mouse) / R (rat)), 0.3 μl Probe Buffer and 0.1μl simethicone oil, make up 1.5μl with Nuclease-free water.

[0062] 1.2 Briefly centrifuge to collect the sample to the bottom of the tube, place the sample in the PCR machine, and operate according to the following procedure, which takes about 15-20 minutes in total.

[0063] 95℃ 2min

[0064] 95-22℃ 0.1℃ / sec

[0065] 22℃ 5min

[0066] Centrifuge briefly to collect the sample at the bottom of the tube, place it on ice, and proceed to the next step immediately.

[0067] 1.3 R...

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Abstract

The invention discloses a construction method and application of a sequencing library of long-chain non-coding RNA. The construction method of the sequencing library of the long-chain non-coding RNA comprises the steps of carrying out rRNA removal treatment on a sample to be detected so as to obtain an rRNA removed product; carrying out reverse transcription treatment on the rRNA removed product to obtain single-stranded DNA (Deoxyribonucleic Acid), wherein a primer for reverse transcription treatment has a sequence as shown in SEQ ID NO: 1; carrying out first amplification treatment on the single-stranded DNA so as to obtain double-stranded DNA; carrying out fragmentation treatment on the double-stranded DNA so as to obtain a DNA fragment; and carrying out second amplification treatment on the DNA fragment, wherein a product of the second amplification forms the sequencing library. According to the method, rRNA is removed through an rRNA removal probe firstly, then reverse transcription is carried out, the influence of a large amount of rRNA on mRNA and lncRNA reverse transcription is avoided, moreover, rRNA is removed through the rRNA removal probe, the rRNA removal rate is high,and the stability of the method is good.

Description

technical field [0001] The invention relates to the field of biotechnology, especially the field of gene sequencing. Specifically, the present invention relates to a method for constructing a sequencing library of long-chain non-coding RNA and its application. More specifically, the present invention relates to a method for constructing a sequencing library of long-chain non-coding RNA, and a method for sequencing long-chain non-coding RNA. Background technique [0002] Genome-wide transcriptome analysis is widely used, and the early method is to obtain RNA from a large number of tissue samples for sequencing. However, this traditional method relies on the overall analysis of the gene expression of millions of cells at a time, which often masks biologically meaningful gene expression differences of some specialized cells in specific tissues. Also, in healthy tissues or diseased tissues such as tumors, certain cells are so rare that they cannot be analyzed by any other tech...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6806C12Q1/6869C40B50/06
Inventor 潘伟业韩典霖陈雪李大为玄兆伶王海良王娟肖飞
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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