ChIL-sequencing

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.{{cite journal | vauthors = Meyer CA, Liu XS | title = Identifying and mitigating bias in next-generation sequencing methods for chromatin biology | journal = Nature Reviews. Genetics | volume = 15 | issue = 11 | pages = 709–21 | date = November 2014 | pmid = 25223782 | doi = 10.1038/nrg3788 | pmc = 4473780 }}{{cite journal | vauthors = Baranello L, Kouzine F, Sanford S, Levens D | title = ChIP bias as a function of cross-linking time | journal = Chromosome Research | volume = 24 | issue = 2 | pages = 175–81 | date = May 2016 | pmid = 26685864 | doi = 10.1007/s10577-015-9509-1 | pmc = 4860130 }} As well, ChIP-seq suffers from suboptimal signal-to-noise ratios and poor resolution.{{cite journal | vauthors = He C, Bonasio R | title = A cut above | journal = eLife | volume = 6 | date = February 2017 | pmid = 28199181 | doi = 10.7554/eLife.25000 | pmc = 5310838 | doi-access = free }} 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.{{cite journal | vauthors = Harada A, Maehara K, Handa T, Arimura Y, Nogami J, Hayashi-Takanawa Y, Shirahige K, Kurumizaka H, Kimura H, Ohkawa Y | title = A chromatin integration labelling method enables epigenomic profiling with lower input | journal = Nature Cell Biology | volume = 21 | date = December 2018 | issue = 2 | pages = 287–296 | pmid = 30532068 | doi = 10.1038/s41556-018-0248-3 | s2cid = 54463772 }}

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-Seq method may be applied to proteins and modifications, including transcription factors, polymerases, structural proteins, protein modifications, and DNA modifications.

There are detailed ChIL-Seq workflows available in an open-access methods repository.{{Cite journal|vauthors=Ohkawa Y, Kimura H, Handa T, Harada A, Maehara K|date=20 Dec 2018|title=Detailed protocol ─ Chromatin Integration labeling|journal=Protocol Exchange|doi-access=free|doi=10.1038/protex.2018.122}}

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.{{cite journal | vauthors = Harada A, Maehara K, Handa T, Arimura Y, Nogami J, Hayashi-Takanawa Y, Shirahige K, Kurumizaka H, Kimura H, Ohkawa Y | title = A chromatin integration labelling method enables epigenomic profiling with lower input | journal = Nature Cell Biology | volume = 21 | date = December 2018 | issue = 2 | pages = 287–296 | pmid = 30532068 | doi = 10.1038/s41556-018-0248-3 | s2cid = 54463772 }} 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. As with other techniques using Tn5, the library preparation has a strong GC bias and has poor sensitivity in low GC regions or genomes with high variance in GC content.{{cite journal |last1=Lan |first1=James H. |last2=Yin |first2=Yuxin |last3=Reed |first3=Elaine F. |last4=Moua |first4=Kevin |last5=Thomas |first5=Kimberly |last6=Zhang |first6=Qiuheng |title=Impact of three Illumina library construction methods on GC bias and HLA genotype calling |journal=Human Immunology |date=March 2015 |volume=76 |issue=2–3 |pages=166–175 |doi=10.1016/j.humimm.2014.12.016 |pmid=25543015 |pmc=5089167 }}{{cite journal |last1=Sato |first1=Mitsuhiko P |last2=Ogura |first2=Yoshitoshi |last3=Nakamura |first3=Keiji |last4=Nishida |first4=Ruriko |last5=Gotoh |first5=Yasuhiro |last6=Hayashi |first6=Masahiro |last7=Hisatsune |first7=Junzo |last8=Sugai |first8=Motoyuki |last9=Takehiko |first9=Itoh |last10=Hayashi |first10=Tetsuya |title=Comparison of the sequencing bias of currently available library preparation kits for Illumina sequencing of bacterial genomes and metagenomes |journal=DNA Research |date=1 October 2019 |volume=26 |issue=5 |pages=391–398 |doi=10.1093/dnares/dsz017 |pmid=31364694 |pmc=6796507 |doi-access=free }}{{cite journal |last1=Chen |first1=Yen-Chun |last2=Liu |first2=Tsunglin |last3=Yu |first3=Chun-Hui |last4=Chiang |first4=Tzen-Yuh |last5=Hwang |first5=Chi-Chuan |title=Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome Assembly |journal=PLOS ONE |date=29 April 2013 |volume=8 |issue=4 |pages=e62856 |doi=10.1371/journal.pone.0062856 |pmid=23638157 |pmc=3639258 |bibcode=2013PLoSO...862856C |doi-access=free }}

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. However, the consumables cost of ChIL-Seq is substantially lower allowing more samples to be processed. {{cite journal |last1=Handa |first1=Tetsuya |last2=Harada |first2=Akihito |last3=Maehara |first3=Kazumitsu |last4=Sato |first4=Shoko |last5=Nakao |first5=Masaru |last6=Goto |first6=Naoki |last7=Kurumizaka |first7=Hitoshi |last8=Ohkawa |first8=Yasuyuki |last9=Kimura |first9=Hiroshi |title=Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input |journal=Nature Protocols |date=October 2020 |volume=15 |issue=10 |pages=3334–3360 |doi=10.1038/s41596-020-0375-8 |pmid=32807906 |s2cid=221145784 |url=https://www.nature.com/articles/s41596-020-0375-8 |access-date=3 December 2020|url-access=subscription }}

• 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.

• ChIP-on-chip
• ChIP-Seq
• CUT&RUN
• CUT&Tag

{{reflist}}

{{DEFAULTSORT:ChIL sequencing}}

Category:DNA sequencing

Category:Protein methods

Category:Molecular biology techniques