Methods for preparing sequencing libraries
Improvements in chromatin immunoprecipitation-high throughput sequencing techniques has allowed the creation of chromatin maps from limited biological sample sizes that cannot be evaluated using conventional chromatin immunoprecipitation-sequencing protocols. For example, a modified universal primer is utilized that incorporates restriction enzymes into chromatin immunoprecipitation fragments before amplification. The improved method allows the sample sizes to be several orders of magnitude less than that required for standard ChIP-Seq techniques.
1. A method comprising:
a) providing;
i) a biological sample comprising less than 50,000 cells, wherein said biological sample comprises genomic DNA;
ii) a hairpin primer comprising at least one restriction enzyme binding site, at least one universal primer sequence ranging between four and thirteen nucleotides in length, and at least one random nucleotide sequence of at least eight nucleotides in length;
iii) a second primer comprising said at least one restriction enzyme binding site sequence;
iv) a restriction enzyme capable of creating a single nucleotide overhang at said restriction enzyme binding site sequences; and
v) a polymerase;
b) fragmenting said genomic DNA wherein a plurality of DNA fragments are created;
c) extending said plurality of DNA fragments with said hairpin primer and said polymerase so as to create a first extension product;
d) amplifying said first extension product with said second primer and said polymerase so as to create a second extension product, wherein the 3′ and 5′ ends of said second extension product each contain said restriction enzyme site sequence;
e) cleaving the 3′ and 5′ ends of said second extension product at said restriction enzyme sites with said restriction enzyme, thereby creating a 3′ cleavage product and a 5′ cleavage product wherein each of said cleavage products are identical and comprise said single nucleotide overhang.
2. The method of claim 1 , wherein said method further comprises step e) attaching a sequencing adapter to each of said cleavage products via said single nucleotide overhang.
3. The method of claim 1 , wherein said genomic DNA fragments are selected from the group consisting of genomic DNA chromatin immunoprecipitated fragments, genomic DNA restriction fragments, and genomic DNA sonication fragments.
4. The method of claim 1 , wherein said random nucleotide sequence comprises between approximately eight to eleven nucleotide residues.
5. The method of claim 1 , wherein said random nucleotide sequence comprises nine nucleotide residues.
6. The method of claim 1 , wherein said polymerase has no 3′→5′ exonuclease activity.
7. The method of claim 1 , wherein said polymerase comprises strand displacement activity.