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Broad Team Tailors ChIP-seq Method for Low Cell Numbers, Limited DNA Volumes

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By Molika Ashford

Researchers at the Broad Institute have published a protocol for performing chromatin immunoprecipitation sequencing on scarce amounts of cells — two to three orders of magnitude fewer cells than are required for standard ChIP-seq — and low volumes of DNA.

Combining a small-scale ChIP-seq assay and a three-stage library preparation method, the Broad coauthors, Mazhar Adli and Bradley Bernstein, created a protocol dubbed nano-ChIP-seq that they used to generate chromatin state maps from as few as 10,000 cultured embryonic stem cells and 25,000 hematopoietic stem cells. They showed that the method works with ChIP DNA volumes in the range of picograms, as opposed to the nanogram volumes that are required for standard ChIP-seq.

In their report, published in the October issue of Nature Protocols, the authors projected that the method should be applicable to other biologically and clinically important cell types as well.

Adli told In Sequence that he and Bernstein developed the protocol to overcome two main limitations in standard ChIP-seq that have prevented researchers from using the method on rare cells or small samples: standard ChIP's need for a large number of cells and standard sequencing library preparation's requirement of a significant volume of DNA.

"Because of these [two limitations], when we want to study biologically important, but more rare cell types – say hematopoietic stem cells or some biopsies where you can't get millions of cells – we were not able to apply this powerful technique," he said. "So we developed this method that we call nano-ChIP sequencing."

Adli and Bernstein's protocol — developed for Illumina technology, but adaptable to other platforms, according to Adli — overcomes each of the two main limitations of standard ChIP-seq, he said.

Using small-scale or "mini" ChIP-seq, he explained, reduces the number of cells required at the head of the procedure, while a new amplification and library preparation method allows for sequencing of a small amount of DNA.

For their small-scale ChIP step, the Broad researchers optimized a previously developed method by tweaking sonication conditions and antibody and bead concentrations.

The library-preparation method is what sets the protocol apart not just from standard ChIP-seq, but also from other methods to adapt ChIP to sequencing small samples, Adli explained.

"DNA yields from small-scale ChIP experiments are typically too low for conventional sequencing library protocols," the authors wrote in the paper, so the team "adapted and optimized a random amplification method previously used for array platforms" that was developed by Pat Brown and colleagues at Stanford.

In the library prep method, ChIP DNA is primed with custom primers, which Adli designed with a restriction enzyme site that results in a final template with a 3'-adenine overhang that can be ligated with standard Illumina adapters.

"The 3'-A is critical, because it means you can seamlessly integrate into the third or fourth step of the Illumina protocol," Adli said. "This is not only saving time, but more importantly saving me material, because in the Illumina library preparation, after each stage you have to purify the DNA and you lose most of the DNA doing these precipitation steps."

In the paper, Adli and Bernstein describe how they applied the protocol to embryonic stem cells as well as hematopoietic stem and progenitor cells isolated via fluorescence activated cell sorting. Adli said that the hematopoietic stem cell experiments, which yielded state maps for three different histone modifications at the whole-genome level, provided insight into the chromatin biology of this cell type for the first time.
"This wasn’t the first protocol we tried and it worked. We tried a lot of different methods and this was the best one," said Adli. "They say necessity is the mother of invention. I really had to develop this protocol."

He noted, however, that nano-ChIP seq adds some complexity to standard ChIP-seq. "It requires some hands-on experience," he said. "But [these] experiments are already complex, so if someone is experienced with normal ChIP-seq, nano-ChIP-seq is not hard at all."

Additionally, he said the cost should be "very comparable" to standard ChIP-seq and may even save a bit in reagent costs since the sequencing library protocol skips some of the steps of standard Illumina library prep.

Adli said that he has patented the library preparation method and the Merck-owned company EMD Millipore is working on commercializing a kit based on the protocol.

Adli is also working on further refining the method to investigate smaller numbers of cells.

"The ultimate aim is to be able to study chromatin in a single cell," he said. "Until we are able to do this, no matter which technology we use, we are basically averaging the population, which masks the behavior of different cells.

"I wouldn't say we are very close to being able to do it, but we have some ideas that we are testing," he said.

Additionally, the two reported in their paper that the amplification procedure alone might also be adapted for sequencing DNA from forensic, archaeological, or other limited samples.

Adli also said several other research groups have contacted him for advice and collaboration in using the method for their research. "We believe that the chromatin field is moving forward at a high speed," he said. "And we believe that people will start being interested in doing more chromatin studies in rare cell types like biopsy samples, for instance."


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