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Index-first chromatin immunoprecipitation (iChIP) high-throughput sequencing experimental method applied to zebrafish embryos

A zebrafish embryo and co-immunoprecipitation technique, applied in the field of high-throughput molecular biology experiments, can solve the problem that chromatin modification is limited to the cellular level

Inactive Publication Date: 2016-04-06
TONGJI UNIV
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Problems solved by technology

[0004] At present, iChIP-seq technology is only used in cell samples, and the research on the dynamic changes of chromatin modification and the relationship with gene expression is only limited to the cell level, and it has not been reported to be applied in zebrafish embryonic cells

Method used

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Experimental program
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Embodiment

[0036] Harvest samples of successfully fertilized zebrafish early embryos. The specific method is as follows:

[0037] (1) Pretreatment of zebrafish embryo samples:

[0038] Get 64 cell stage, 256 cell stage, 1k cell stage, Oblong cell stage, Dome / 30% epiboly each 200 embryos in 2ml centrifuge tube, add 1% formaldehyde 1750ul (here 1% The formaldehyde is: 270ul of 37% formaldehyde plus 4.73ml of PBS (diluted with 4.73ml PBS) for cross-linking on a shaker for 3 minutes, then add 200ul of 1.25M glycine and shake for 5 minutes to stop the cross-linking.

[0039] (2) Nuclear lysis of zebrafish embryos:

[0040] The pretreated zebrafish embryo samples were washed three times with PBS plus protein inhibitors, and the liquid was aspirated as clean as possible for the last time. Add 1ml of pre-cooled lysis buffer 10mM Tris-HCl pH7.5, 10mMNaCl, 0.5% NP-40 (plus protein inhibitor), pipette the embryo repeatedly on ice with a 1ml wide-mouth pipette until the embryo is lysed, and place...

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Abstract

The invention relates to an index-first chromatin immunoprecipitation (iChIP) high-throughput sequencing (iChIP-seq) experimental method applied to zebrafish embryos. The method comprises the following steps: firstly, performing formaldehyde crosslinking on zebrafish embryo samples; secondly, acquiring mononucleosome through MNase (Micrococcal Nuclease) enzyme digestion; fixing chromatin on magnetic beads through H3 antigens; labeling various different samples on the magnetic beads with index joints; releasing the chromatin from the magnetic beads, concentrating, mixing the samples together, performing a chromatin immunoprecipitation reaction on various antigens to obtain DNA (Deoxyribose Nucleic Acid) sample, and purifying the DNA sample to perform library establishment and sequencing. The iChIP is an improvement on conventional ChIP (Chromatin Immunoprecipitation). According to the method, each sample is marked before ChIP, and then all the samples are mixed together to perform an experiment, so that a dynamical change decorated by the chromatin in a developing process can be observed, experimental deviations possibly caused by a small quantity of early-period cells are reduced, and the chromatin-modified dynamical change in the whole developing process can be quantitatively observed through the quantity of cells.

Description

technical field [0001] The invention belongs to the technical field of high-throughput experiments of molecular biology, and is mainly used for studying dynamic changes of genome-wide chromatin modification and related gene transcription mechanisms in the early embryo development process of zebrafish. Background technique [0002] Embryo development has always been one of the subjects of life science research. The development from a single cell to a multicellular individual is the result of the sequential expression of many genes and proteins in time and space. Among them, chromatin modification plays an important role in embryonic development, but there are relatively few studies on its dynamic changes during development. Zebrafish provides a good model for the development of organisms, but experimental technical limitations hinder Excellent study of the dynamics of chromatin modification at various stages of development. Although traditional chromatin immunoprecipitation...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6888C12N15/1003C12N2330/31C12Q1/6869
Inventor 刘桂芬张勇陈韵如胡圣恩曾诗扬陈晓兰
Owner TONGJI UNIV
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