ChIL-sequencing

ChIL sequencing (ChIL-seq), also known as Chromatin Integration Labeling sequencing, is a method used to analyze protein interactions with DNA. ChIL-sequencing combines antibody-targeted controlled cleavage by Tn5 transposase with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that ChIL-Sequencing does not. ChIL-Seq is a precise technique that reduces sample loss could be applied to single-cells. [1]

Uses

ChIL-sequencing can be used to examine gene regulation or to analyze transcription factor and other chromatin-associated protein binding. Protein-DNA interactions regulate gene expression and are responsible for many biological processes and disease states. This epigenetic information is complementary to genotype and expression analysis. ChIL-Seq is an alternative to the current standard of ChIP-seq. ChIP-Seq suffers from limitations due to the cross linking step in ChIP-Seq protocols that can promote epitope masking and generate false-positive binding sites.[2][3] As well, ChIP-seq suffers from suboptimal signal-to-noise ratios and poor resolution.[4] ChIL-sequencing has the advantage of being a simpler technique suitable for low sample input due to the high signal-to-noise ratio, requiring less depth in sequencing for higher sensitivity.[5]

Specific DNA sites in direct physical interaction with proteins such as transcription factors can be isolated by Protein-A (pA) conjugated Tn5 bound to a protein of interest. Tn5 mediated cleavage produces a library of target DNA sites bound to a protein of interest in situ. Sequencing of prepared DNA libraries and comparison to whole-genome sequence databases allows researchers to analyze the interactions between target proteins and DNA, as well as differences in epigenetic chromatin modifications. Therefore, the ChIL-Se method may be applied to proteins and modifications, including transcription factors, polymerases, structural proteins, protein modifications, and DNA modifications.

Protocols

There are detailed ChIL-Seq workflows available in an open-access methods repository.[6]

Limitations

The primary limitation of ChIL-seq is the likelihood of over-digestion of DNA due to inappropriate timing of the Magnesium-dependent Tn5 reaction. This is biased towards open chromatin like ATAC-Seq and similar techniques [5]. A similar limitation exists for contemporary ChIP-Seq protocols where enzymatic or sonicated DNA shearing must be optimized. As with ChIP-Seq, a good quality antibody targeting the protein of interest is required.

ChIL-Seq requires numerous laboratory steps and takes longer than other techniques such as CUT&RUN or CUT&Tag. It is still a broadly applicable technique which avoids sample loss suitable for small numbers of cells.

Similar methods
  • Sono-Seq: Identical to ChIP-Seq but without the immunoprecipitation step.
  • HITS-CLIP: Also called CLIP-Seq, employed to detect interactions with RNA rather than DNA.
  • PAR-CLIP: A method for identifying the binding sites of cellular RNA-binding proteins.
  • RIP-Chip: Similar to ChIP-Seq, but does not employ cross linking methods and utilizes microarray analysis instead of sequencing.
  • SELEX: Employed to determine consensus binding sequences.
  • Competition-ChIP: Measures relative replacement dynamics on DNA.
  • ChiRP-Seq: Measures RNA-bound DNA and proteins.
  • ChIP-exo: Employs exonuclease treatment to achieve up to single base-pair resolution
  • ChIP-nexus: Potential improvement on ChIP-exo, capable of achieving up to single base-pair resolution.
  • DRIP-seq: Employs S9.6 antibody to precipitate three-stranded DND:RNA hybrids called R-loops.
  • TCP-seq: Principally similar method to measure mRNA translation dynamics.
  • DamID: Uses enrichment of methylated DNA sequences to detect protein-DNA interaction without antibodies.
gollark: Are you running them on Linux or OpenBSD or what?
gollark: I'm working on apiomemetics 2.0™.
gollark: Well, yes, but that's boring.
gollark: There's some weirdness when I [REDACTED] which looks like [REDACTED] file descriptor stuff.
gollark: Idea: macro for arbitrarily d-including cadr?

See also

References
  1. "When less is more: A promising approach for low-cell-number epigenomic profiling". Science Daily. 11 December 2018. Retrieved 24 December 2019.
  2. Meyer CA, Liu XS (November 2014). "Identifying and mitigating bias in next-generation sequencing methods for chromatin biology". Nature Reviews. Genetics. 15 (11): 709–21. doi:10.1038/nrg3788. PMC 4473780. PMID 25223782.
  3. Baranello L, Kouzine F, Sanford S, Levens D (May 2016). "ChIP bias as a function of cross-linking time". Chromosome Research. 24 (2): 175–81. doi:10.1007/s10577-015-9509-1. PMC 4860130. PMID 26685864.
  4. He C, Bonasio R (February 2017). "A cut above". eLife. 6. doi:10.7554/eLife.25000. PMC 5310838. PMID 28199181.
  5. Harada A, Maehara K, Handa T, Arimura Y, Nogami J, Hayashi-Takanawa Y, Shirahige K, Kurumizaka H, Kimura H, Ohkawa Y (December 2018). "A chromatin integration labelling method enables epigenomic profiling with lower input". Nature Cell Biology. 21. doi:10.1038/s41556-018-0248-3. PMID 30532068.
  6. Ohkawa Y, Kimura H, Handa T, Harada A, Maehara K (20 Dec 2018). "Detailed protocol ─ Chromatin Integration labeling". Protocol Exchange. doi:10.1038/protex.2018.122.
This article is issued from Wikipedia. The text is licensed under Creative Commons - Attribution - Sharealike. Additional terms may apply for the media files.