Muscle tissue extraction method for separating bovine muscle stem cells and application of muscle tissue extraction method
A technology of muscle tissue and extraction method, which is applied in the field of cell engineering, can solve problems that have yet to be revealed, and the openness of chromatin has yet to be revealed, and achieve the effect of reducing costs
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Embodiment 1
[0045] Example 1 Establishment of an in vitro isolation and culture system for bovine muscle stem cells
[0046] In the examples of the present invention, the formula of 50 mL of type I collagenase solution (0.2%) used is shown in Table 1.
[0047] Table 1 50mL type I collagenase solution (0.2%) formula
[0048] A low-glucose DMEM basal medium 39mL (sugar content: 1.0g / L) a neutral enzyme II 10mL (final concentration: 1U / mL) A collagenase I 1mL (final concentration: 0.1mg / mL)
[0049] In the examples of the present invention, the formula of 50 mL of pancreatin solution (0.25%) used is shown in Table 2.
[0050] Table 2 50mL pancreatin solution (0.25%) formula
[0051] Phosphate buffer 39mL Pancreatin 10mL (final concentration: 1U / mL)
[0052] In the implementation of the present invention, the formulation of 50 mL of bovine muscle stem cell primary culture medium used is shown in Table 3.
[0053] Table 3 Formulation of ...
Embodiment 2
[0086] Example 2 Construction of bovine muscle stem cell super-enhancer method
[0087] In the implementation of the present invention, chromatin co-immunoprecipitation high-throughput sequencing is performed based on muscle stem cells to obtain enhancer markers constituting different chromosomes in the whole genome, and the specific steps are as follows:
[0088] (1) Acquisition of raw data constituting enhancers
[0089] ①Use antibodies specific for the methylation of the fourth lysine residue of histone H3 (referred to as H3K4me1) and the acetylation of the 27th lysine residue of histone H3 (referred to as H3K27ac) to stain the target protein of the cell sample Co-immunoprecipitation reaction was performed to obtain genomic DNA fragments bound by H3K4me1 and H3K27ac tagged proteins; ② Purify and enrich the DNA fragments respectively; ③ Perform high-throughput sequencing on the DNA fragment products respectively; The base calling analysis is converted into a sequence file (...
Embodiment 3
[0117] Example 3 Construction of super-enhancer map of bovine muscle stem cells
[0118] In the embodiment of the present invention, to construct a super-enhancer map of bovine muscle stem cells, the specific steps are:
[0119] (1) Identification of super-enhancers
[0120] ① Based on the ROSE algorithm, the H3K27ac signal value of each constituting enhancer was calculated for the enhancer binding sites identified by H3K27ac in each enhancer region on the genome;
[0121] ② Calculate the Med1 signal of each enhancer region according to the H3K27ac signal value that constitutes the enhancer;
[0122] ③Order the Med1 signal in the enhancer region, represented by the coordinate axis. For example, the Med1 enrichment on the y-axis is arranged along the x-axis, and a graph is drawn to obtain a graph. The signal value obtained at the tangent point of the tangent line with a slope of 1 on the curve is used to distinguish between super-strong enhancers and ordinary enhancers. Thresh...
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