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A technology of co-immunoprecipitation and chromatin, applied in the field of sperm chromatin co-immunoprecipitation, can solve problems such as low efficiency, and achieve the effect of reducing difficulty, improving digestion efficiency and improving efficiency
Pending Publication Date: 2021-11-16
上海市生物医药技术研究院
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[0004] However, due to the small amount of histones contained in sperm (about 6-15%), the efficiency of c
Method used
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Embodiment 1
[0056] 1. Take human sperm (the number is about 2×10) frozen at -80°C 7 ), adding paraformaldehyde (PFA) with a mass concentration of 0.5%, and fixing at 22° C. for 10 min.
[0057] 2. Centrifuge at 5,000g at 22°C for 10 minutes, and discard the supernatant.
[0058] 3. Add 4°C pre-cooled PBS to wash once, centrifuge at 5,000g at 22°C for 10 minutes, and discard the supernatant.
[0059] 4. Add 300 μl of lysis solution (0.5% SDS, 10 mM DTT) to the sperm pellet, and incubate at 22° C. for 1 hr.
[0060] 5. Then add 3 μl of 1M N-ethylmaleimide to terminate the effect of DTT.
[0061] 6. Then use ultrasonic treatment, ultrasonic 30s (choose Low power of the ultrasonic device), interval 45s, 10 cycles; use micrococcal nuclease to digest after ultrasonication, the enzyme addition is 200U / 10 6 spermatozoa, the enzymolysis temperature was 37°C, and the enzymolysis time was 30 minutes to obtain digestive juice.
[0062] 7. Centrifuge the above digestive solution at 12,000rpm for 1...
Embodiment 2
[0098] Human sperm was replaced with mouse sperm, and the rest of the operations were the same as in Example 1.
[0099] Validation of H3K4me2 and H3K27me3 positive and negative sites as Figure 4 , Figure 5 shown.
Embodiment 3
[0101] Optimize the ultrasonic conditions, change the number of ultrasonic cycles to 0, 3, 6, and 10, and then perform agarose gel electrophoresis on the supernatant and precipitate of the digestive solution. The results are as follows: Image 6 shown.
[0102] The supernatant and precipitate after digestion and co-immunoprecipitation under the above conditions were used to calculate the DNA yield, and the results are shown in Table 2.
[0103] Table 2 DNA yield after optimization of ultrasonic conditions
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Abstract
The invention discloses a sperm chromatin co-immunoprecipitation method. The method belongs to the field of target nucleic acid purification and next-generation sequencing library building sample pretreatment. The method includes the following steps: sperm digestion: subjecting the sperms to paraformaldehyde immobilization, cleaning and split, digesting the sperms by adopting a micrococcus nuclease digestion-ultrasonic combined method to obtain digestive juice; ultrasonic parameters are as follows: performing ultrasonic treatment for 30 seconds by the interval of 45 seconds for 10 cycles; setting the digestion parameters of the micrococcus nuclease are as follows: the enzyme addition amount is 200U/106 sperms, the enzymolysis temperature is 37 DEG C, and the enzymolysis time is 30 minutes; chromatin separation: performing preliminary cleaning on the digestive juice to obtain chromatin; and chromatin co-immunoprecipitation: forming a compound by the antibody and protein A agrose, combining the compound with a corresponding antigen in chromatin, and then cleaning and eluting to obtain a co-immunoprecipitation reaction solution. According to the method, ultrasonic and enzyme digestion are combined, so that the digestion efficiency is improved, the DNA recovery rate is increased, the library building difficulty is reduced, and the sperm co-immunoprecipitation efficiency is improved.
Description
technical field [0001] The invention relates to the technical field of targeted nucleic acid purification and sample pretreatment for next-generation sequencing library construction, and more specifically relates to a sperm chromatin immunoprecipitation method. Background technique [0002] At present, there are many methods for studying the interaction between proteins, such as yeast two-hybrid elution, co-immunoprecipitation, and pull-down. Among them, co-immunoprecipitation is a relatively stable and convenient method, and it is also a method that has been widely used in the scientific research of proteome. [0003] Co-immunoprecipitation is a technique used to study protein interactions based on specific binding between antibodies and antigens. The intact cells are mainly lysed with non-denaturing agents, which maintains the interaction between many proteins in the cell and facilitates the detection of the interaction between proteins. The antibody against protein A fo...
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