Generating a Three-Dimensional Genome from Xenopus with Hi-C
| Autor: | Sven Heinz, Ian K. Quigley |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0303 health sciences
Chromosome Genome Molecular biology General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound Restriction enzyme Restriction site genomic DNA 0302 clinical medicine chemistry Genomic library 030217 neurology & neurosurgery DNA 030304 developmental biology Reference genome |
| Zdroj: | Cold Spring Harbor Protocols. 2019:pdb.prot098343 |
| ISSN: | 1559-6095 1940-3402 |
| DOI: | 10.1101/pdb.prot098343 |
| Popis: | Hi-C is a sequencing-based method that captures three-dimensional (3-D) genome interactions by counting the interaction frequencies of pairs of genomic loci. This protocol describes the application of in situ Hi-C to the Xenopus embryo. Briefly, after fixing embryos with formaldehyde, nuclei are isolated and chromatin is digested with a restriction enzyme. Restriction sites are filled in with a biotinylated nucleotide and the blunted ends are re-ligated in place, all while still contained in the nuclei (i.e., in situ). Subsequently, the re-ligated genomic DNA is isolated and fragmented by sonication. Biotinylated ligation junctions are captured with streptavidin-coated beads, and DNA fragments are amplified by ligation-mediated polymerase chain reaction (LM-PCR). The PCR product is isolated and sequenced from both ends (paired-end), and informatics methods are then applied to align the two sides of the ligation junctions to the reference genome. Because ligation occurs much more frequently intra- than interchromosomally, and with generally decreasing frequency the further away DNA loci are from each other on the linear chromosome, interaction frequency information can be used to assist in assembling genomes and to phase haplotypes, which is especially useful in the case of a tetraploid organism such as X. laevis. Our streamlined version of in situ Hi-C was optimized for high throughput and low cost, and enables generation of high-quality Hi-C libraries from small cell numbers (down to ∼10,000 cells) in 2 d. |
| Databáze: | OpenAIRE |
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