Trace cell ChIP (Chromatin Immunoprecipitation) method

Abstract

The invention relates to a trace cell ChIP (Chromatin Immunoprecipitation) method. The method comprises the following steps: 1) dividing a cell sample to be detected into n groups, wherein n is a natural number which is not equal to 0; 2) carrying out crosslinking fixation on the nth group sample; 3) treating the nth group sample by utilizing a cell membrane perforating agent; 4) carrying out enzyme digestion by utilizing Tn5 transposase and breaking a chromatin segment of the nth group sample, and connecting a barcode sequence and a primer sequence to two ends of a production segment DNA (Deoxyribonucleic Acid), wherein when the n is greater than or equal to 2, the barcode sequence and / or the primer sequence used in each group of step 4) are / is different; 5) when the n is greater than orequal to 2, combining samples of all groups; 6) enriching a DNA segment combined with target protein and taking a primer as an index for creating a database; sequencing and analyzing. When the methodis used for sequencing, the database creating efficiency is high; the capturing of transcription factor sites of an extremely trace amount of cells can be realized; relatively good efficiency is ensured and the stability and the accuracy are relatively good.

Description

technical field [0001] The invention relates to the technical field of molecular biology experiments, in particular to a micro-cell ChIP method. Background technique [0002] The gene regulation and expression of organisms is an extremely complex but orderly process. The genome DNA of organisms exists in the form of chromatin in cells, and the interaction between proteins and DNA is an important basis for cells to perform functions. Therefore, studying the interaction of proteins and DNA in the context of chromatin can further understand gene expression and its regulatory patterns. [0003] Chromatin Immunoprecipitation (ChIP) is a standard method for studying DNA-protein interactions in vivo. Chromatin immunoprecipitation technology can locate and analyze the interaction sites of proteins and DNA in vivo, and combine ChIP technology with other methods, such as high-density chip (microarray), sequencing (sequencing), in vivo footprinting and other technologies to obtain Th...

AI Technical Summary

Problems solved by technology

The main reason is that in the traditional Illumina TruSeq library construction strategy, the adapter and DNA fragment connection efficiency is low, so that many fragments are not enriched and lost in the subsequent amplification set

Especially when the research problem needs to be aimed at a small number of cell populations, serious information loss prevents the experimenter from obtaining more accurate real information

[0008] 2. The method of breaking chromatin fragments by ultrasound is not conducive to the study of transcription factors that indirectly bind to DNA

Even for formaldehyde-crosslinked samples, this indirect bond is relatively easy to destroy

The ultrasonic process has a certain degree of physical crushing strength, which is easy to interfere with the stability of these indirect binding target proteins and DNA binding, resulting in the loss of subsequent information

[0010] 3. The library has high non-specific background and low detection resolution

These non-specific sites will be preserved in the subsequent library construction and sequencing process, which will become the interference background of the target protein map analysis, obtain broad peaks, and reduce the resolution and accuracy of detecting binding sites

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