Methods of genome sequencing and epigenetic analysis
AssigneeCarnegie Institution of Washington
InventorsYixian ZHENG, Xiaobin ZHENG, Sibiao YUE
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Abstract
Methods of ChIP-seq are disclosed herein. These methods of ChIP-seq employ carrier DNA to prevent loss of DNA samples. The greater DNA yields achieved by the presently disclosed technology permit ChIP-seq of a small number of cells, permitting epigenetic analysis of primary cells of limited quantity.
Claims31
A method of sequencing genomic DNA from a sample of cells, the method comprising:
- (a) fragmenting chromatin in the sample of cells by fixing proteins and nucleic acids of the chromatin, enzymatic digestion of the fixed chromatin and sonication of the sample of cells comprising the enzymatically digested chromatin to produce fragmented chromatin,
- (b) adding a carrier DNA to the fragmented chromatin, wherein the carrier DNA is 5′ biotinylated DNA (“DNA1”), or DNA2)
- (c) precipitating the mixture of carrier DNA and fragmented chromatin,
- (d) annealing a blocking primer that is complementary to the DNA1 to the precipitated mixture to produce a second mixture,
- (e) amplifying the genomic DNA in the second mixture, and
- (f) sequencing the amplified DNA;
- wherein the sample of cells comprise less than 5,000 cells, and
- wherein the blocking primers prevent amplification of the DNA1.
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A method of sequencing genomic DNA from a sample of cells, the method comprising:
- (a) fragmenting chromatin in the sample of cells by fixing proteins and nucleic acids of the chromatin, enzymatic digestion of the fixed chromatin and sonication of the sample of cells comprising the enzymatically digested chromatin to release the fragmented chromatin from the nucleus,
- (b) adding a carrier DNA to the fragmented chromatin of each sample of cells, wherein the carrier DNA is 5′ biotinylated with a 5′ overhang and a 3′ Spacer 3 modification (“DNA2”) or 5′ biotinylated DNA1 without the modifications (DNA1).
- (c) precipitating the mixture of carrier DNA and fragmented chromatin and purifying the DNA,
- (d) amplifying the purified DNA and sequencing the amplified DNA,
- wherein the sample of cells comprise between 1 and 20,000 cells.
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A method of sequencing genomic DNA from a sample of cells, the method comprising:
- (a) combining a sample of cells of interest with a sample of bulking cells,
- (b) fragmenting chromatin in the combined sample of cells of interest and sample of bulking cells by fixing proteins and nucleic acids of the chromatin, enzymatic digestion of the fixed chromatin and sonication of the sample of cells comprising the enzymatically digested chromatin to produce fragmented chromatin,
- (c) precipitating the fragmented chromatin of the cells of interest,
- (d) amplifying the genomic DNA from the sample of cells, and
- (e) sequencing the amplified DNA;
- wherein the sample of cells comprise between 1 and 20,000 cells, and
- wherein the bulking cells are yeast cells or E. coli cells.
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Dependencies are inferred from the claim text.