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Library construction method based on promoter and library thereof

A promoter and library technology, applied in the field of gene sequencing, can solve the problems of inapplicability and low coverage of single-cell methylation sequencing, and achieve the effect of improving the coverage.

Pending Publication Date: 2022-04-29
BGI GENOMICS CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, this TET1 is not suitable for most laboratories
In summary, the coverage of single-cell methylation-sequencing is low, reaching only 20% of CpGs and less than 10% of the genome

Method used

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  • Library construction method based on promoter and library thereof
  • Library construction method based on promoter and library thereof
  • Library construction method based on promoter and library thereof

Examples

Experimental program
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Embodiment 1

[0090] figure 1 It is a schematic diagram of the database construction process in this embodiment.

[0091] In this example, a 100 pg DNA methylation library was constructed.

[0092] Specific steps are as follows:

[0093] 1. Annealing of oligo (New Tn5-pho) and oligo (New bio-bottom) to form a double-stranded linker, and then embedding with transposase Tn5 to form a complex. "oligo" refers to an oligonucleotide, "pho" refers to a phosphate group, and "bio-bottom" refers to an oligonucleotide modified with biotin.

[0094] The nucleotides used in this example are shown in Table 2.

[0095] Table 2

[0096]

[0097] "N" represents a random base, which is any one of A, T, C, and G.

[0098] 2. Tn5 interrupts genomic DNA

[0099] Take 100pg of DNA and interrupt it with the Tn5 enzyme embedded in the linker. The interrupted reaction system is 10 μL, in which the amount of transposase used is 0.5 μL, and the amount of 5XtagH buffer used is 2 μL. React on a PCR instrument at...

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Abstract

The invention discloses a method for constructing a library based on a promoter and the library. The method comprises the following steps: a breaking step: breaking a to-be-detected sample to obtain a broken to-be-detected sample; carrying out enzymatic methylation conversion, namely carrying out enzymatic methylation conversion on the broken sample to be detected; connecting a T7 promoter, namely annealing the to-be-detected sample subjected to the enzymatic methylation conversion treatment and the T7 promoter, and reacting to obtain a to-be-detected sample connected with the T7 promoter; in-vitro transcription: transcribing the to-be-detected sample connected with the T7 promoter into RNA (Ribonucleic Acid); and reverse transcription and amplification: carrying out reverse transcription and PCR amplification on the RNA to obtain the library. Compared with direct PCR (polymerase chain reaction) in the prior art, transcription (aiming at DNA (deoxyribonucleic acid) rich in uracil) of the T7 is more uniform, more RNA (ribonucleic acid) can be obtained by transcription to serve as a template for next cDNA amplification, and methylation coverage is greatly improved.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a method for constructing a library based on a promoter and the library thereof. Background technique [0002] In recent years, the rapid development of single-cell sequencing technologies has provided many valuable insights into complex biological systems, for example, revealing complex and rare cell populations and tracking the developmental trajectories of different cell lineages. There are many methods for single-cell sequencing, the most popular of which is conventional single-cell RNA or DNA sequencing. In addition to RNA and DNA information, epigenetic modification refers to changes in genetic material that cause heritable phenotypes without changing the DNA sequence itself. Among these epigenetic modifications, 5-methylcytosine (5-methylcytosine, 5mC) is the most abundant modifier in vertebrates and has become an important factor in the regulation of embryonic de...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12Q1/6806C12Q1/6869
CPCC40B50/06C40B40/06C12Q1/6806C12Q1/6869
Inventor 王娟唐冲
Owner BGI GENOMICS CO LTD
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