Method for constructing DNA sequencing library of genome under detection and application of method

A DNA sequencing and sequencing library technology, applied in the biological field, can solve the problems of limited antibody quality and inability to obtain effective histone modification information, so as to avoid local damage and improve recognition efficiency

Active Publication Date: 2016-07-13
TSINGHUA UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, chromatin immunoprecipitation technology is extremely limited by many factors such as the number of cells and the quality of antibodies. If a sufficient amount of DNA for library construction cannot be obtained, effective histone modification information cannot be obtained.
The traditional chromatin immunoprecipitation technique includes several important steps such as crosslin

Method used

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  • Method for constructing DNA sequencing library of genome under detection and application of method
  • Method for constructing DNA sequencing library of genome under detection and application of method
  • Method for constructing DNA sequencing library of genome under detection and application of method

Examples

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Example Embodiment

[0088] Example 1 Construction of DNA sequencing library of cellular genome

[0089] 1.1 Reagent preparation

[0090] Lysisbuffer

[0091] 0.5% NP-40

[0092] 0.5% Tween

[0093] 0.1% SDS

[0094] MNase working buffer (MNaseworkingbuffer)

[0095] 100mM Tris-HClph8.0

[0096] 2mMCaCl2

[0097] MNase Dilutebuffer

[0098] 50mM Tris‐HCl, pH 8.0

[0099] 1mM CaCl2

[0100] 0.2% TritonX‐100

[0101] 10xMNase Stop Buffer (10xMNaseStopbuffer)

[0102] 110mM Tris‐HCl pH8.0

[0103] 55mM EDTA

[0104] 2xRIPA buffer 2xRIPAbuffer (onice)

[0105] 280mM NaCl

[0106] 1% TritonX‐100

[0107] 0.1% SDS

[0108] 0.2% Na‐Deoxycholate

[0109] 5mM EGTA

[0110] RIPA buffer RIPAbuffer

[0111] 10mM Tris pH 8.0

[0112] 1mM EDTA

[0113] 140mM NaCl

[0114] 1% TritonX‐100

[0115] 0.1% SDS

[0116] 0.1% Na‐Deoxycholate

[0117] Lithium chloride buffer (LiClwashbuffer)

[0118] 250mM LiCl

[0119] 10mM Tris pH 8.0

[0120] 1mM EDTA

[0121] 0.5% NP‐40 (now known as Ige...

Example Embodiment

[0137] Example 2 Screening of dephosphorylating enzymes

[0138] In order to obtain the best dephosphorylation end repair results and ensure the final DNA yield, the inventors screened the most suitable dephosphorylation enzymes from three combinations of T4PNK, T4 DNA polymerase, Klenow or T4PNK or rSAP. The specific experimental process is as follows Said:

[0139] The mouse embryonic stem cells grown on the six-well cell culture plate were taken out of the incubator, the medium was aspirated, and the medium was gently washed with 1 ml of PBS to remove the residual medium. Afterwards, 0.5 ml of 0.05% trypsin was added, digested at 37° for three minutes, and the digestion reaction was terminated with 1 ml of fresh medium. In order to make the cells completely into a single suspension cell state, pipet repeatedly with a large 1ml pipette tip until there are no cell clumps visible to the naked eye. Then centrifuge at low speed at 2000rmp for 5 minutes. After aspirating the s...

Example Embodiment

[0158] Example 3

[0159] In order to verify the validity of the library method proposed in this application, the inventors adopted the most stringent testing method: DNA sequencing technology based on co-immunoprecipitation with a large number of (1x10e7) mouse embryonic stem cells H3K4me3, which is regarded as the gold standard in the ENCODE database (ChIP-sequence) results are compared to see the correlation between the two.

[0160] Specifically, the inventor compared the map data obtained by the inventor and the ENCODE map data, and loaded them into the UCSCbrowser, and then by observing the peak distribution of H3K4me3, combined with its positional relationship with the gene promoter, to determine the Whether the proposed database building method is successful.

[0161] The result is as Figure 16 As can be seen from the results (genomic location: chr6:51,271,690-52,394,589), both the results obtained from millions of cells (ENCODE) and the inventors' ChIP-sequence res...

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Abstract

The invention provides a method for constructing a DNA sequencing library of a genome under detection, comprising the steps: (1), subjecting the genome under detection to first digestion by using micrococcal nuclease to obtain a first digestion product; (2), subjecting the first digestion product to co-immunoprecipitation to obtain a co-immunoprecipitation product; (3), subjecting the co-immunoprecipitation product to second digestion by using protease K to obtain second digestion product; (4), subjecting the second digestion product directly to dephosphorylation by using shrimp alkaline phosphatase to obtain a dephosphorylation product; (5), subjecting the dephosphorylation product directly to denaturalization to obtain a denaturalization product with single-branched DNA molecules; and (6), acquiring the sequencing library by TELP (tailing, extension, ligation and PCR) method based on the denaturalization product with single-branched DNA molecules.

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Claims

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Application Information

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Owner TSINGHUA UNIV
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