Evolution of Epigenetic Research Methods
As evolution progressed, genetic alterations accumulated and a mechanism for gene selection developed. This mechanism is called epigenetics. Since the 1970s many approaches have been developed to investigate epigenetic relations.
A wide variety of illnesses, behaviors, and other health indicators have some level of evidence linking them with epigenetic mechanisms, including cancers of almost all types, cognitive dysfunction, and respiratory, cardiovascular, reproductive, autoimmune, and neurobehavioral illnesses. However, research in this field is still in its infancy and subject to limitations.
In the past five years, and especially in the past year or two, several groundbreaking studies have focused fresh attention on epigenetics. They feature new approaches like CUT&RUN or CUT&Tag emerge which help to enhance the understanding of epigenetics and epigenomics. These techniques offer improvements in high-throughput technologies, analytical techniques, computational capability, mechanistic studies, and bioinformatic strategies. Now previously unthinkable experiments are possible and can be carried out at increased pacing.
Discover our interactive timeline of Epigenetic Research Methods: From ChIC and ChIP-seq to CUT&RUN and CUT&tag. A Chronological listing of applications to research epigenetic correlations based on the first mention in a publication. Click on an application for more information along with matching antibodies, proteins and kits.
2004: ChIC
ChIC (chromatin immunocleavage) consists of tethering the fusion protein pA-MN (protein A fused to micrococcal nuclease) to antibodies that in turn are specifically bound to a chromatin protein.
2006: MNase-seq
MNase-seq, short for micrococcal nuclease digestion with deep sequencing, relies on the use of the non-specific endo-exonuclease micrococcal nuclease, to bind and cleave protein-unbound regions of DNA on chromatin. The uncut DNA is then purified from the proteins and sequenced through one or more of the various Next-Generation sequencing (NGS) methods.
2007: ChIP-seq
ChIP-sequencing, also known as ChIP-seq, combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest.
2010: FAIRE-seq
FAIRE-Seq stands for Formaldehyde-Assisted Isolation of Regulatory Elements. The FAIRE-Seq protocol doesn't require the permeabilization of cells or isolation of nuclei, and can analyse any cell type. It is followed by non-directed chromatin fragmentation and subssequent NGS.
2013: ATAC-seq
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) identifies accessible DNA regions by probing open chromatin with hyperactive mutant Tn5 Transposase that inserts sequencing adapters into open regions of the genome. It is an alternative advanced method for MNase-seq, FAIRE-Seq and DNase-Seq providing increased sensitivity.
2017: CUT&RUN
CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP.
Evolution of Epigenetic Research Methods: Chronological listing of applications to research epigenetic correlations based on the first mention in a publication. Click on an application for more information along with matching antibodies, proteins and kits.
2018: AutoCUT&RUN
AutoCUT&RUN enables high-throughput in situ profiling of chromatin proteins. It utilizes a CUT&RUN protocol adaption with a Beckman Biomek FX liquid handling robot for magnetic separation and temperature control. DNA ends in chromatin fragments are polished for direct ligation of Illumina library adapters.
2019 CUT&Tag
Cleavage under targets and tagmentation (CUT&Tag), combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.
2019: uli.CUT&RUN
Ultra-low input CUT&RUN (uliCUT&RUN) enables interrogation of transcription factor binding from rare cell populations including single cells and individual pre-implantation embryos.
2021: scCUT&Tag
Single-cell CUT&Tag (scCUT&Tag) combines the CUT&Tag technology with droplet-based single-cell library preparation. Enables analysis of histone modifications and transcription factor occupancy at single-cell resolution.
2022: CUT&RUN LoV-U
CUT&RUN Low Volume and Urea (CUT&RUN Lov-U) enables characterization of rare transcription factors, transient interactions, or proteins in complexes that are not directly associated with DNA, while avoiding crosslinking. These targets are difficult to characterize with standard CUT&RUN.
2022: CUT&Tag2for1
CUT&Tag2for1 is a modified method for simultaneous profiling of the accessible and silenced regulome in single cells.
2022: MulTI-Tag
Multiple Target Identification by Tagmentation (MulTI-Tag) is an antibody barcoding approach for profiling multiple chromatin features simultaneously in single cells. The protocol retains high sensitivity and specificity and was able to detect three histone modifications in the same cell.
2022: RT-Tag
Reverse Transcribe and Tagment (RT&Tag) was developed to map chromatin-associated RNA. A chromatin epitope is targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tagmented by Tn5 transposases for downstream sequencing.
References
- : "ChIC and ChEC; genomic mapping of chromatin proteins." in: Molecular cell, Vol. 16, Issue 1, pp. 147-57, (2004) (PubMed).
- : "Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin." in: Genome research, Vol. 16, Issue 12, pp. 1505-16, (2007) (PubMed).
- : "Genome-wide mapping of in vivo protein-DNA interactions." in: Science (New York, N.Y.), Vol. 316, Issue 5830, pp. 1497-502, (2007) (PubMed).
- : "A map of open chromatin in human pancreatic islets." in: Nature genetics, Vol. 42, Issue 3, pp. 255-9, (2010) (PubMed).
- : "Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position." in: Nature methods, Vol. 10, Issue 12, pp. 1213-8, (2014) (PubMed).
- : "A computerised medical manpower database--an assessment of its value in medical education." in: Health bulletin, Vol. 47, Issue 4, pp. 168-75, (1989) (PubMed).
- : "Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs." in: Epigenetics & chromatin, Vol. 11, Issue 1, pp. 74, (2019) (PubMed).
- : "CUT&Tag for efficient epigenomic profiling of small samples and single cells." in: Nature communications, Vol. 10, Issue 1, pp. 1930, (2019) (PubMed).
- : "Profiling of Pluripotency Factors in Single Cells and Early Embryos." in: Cell, Vol. 177, Issue 5, pp. 1319-1329.e11, (2020) (PubMed).
- : "Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues." in: Nature biotechnology, Vol. 39, Issue 7, pp. 825-835, (2021) (PubMed).
- : "CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells." in: , Vol. 23, Issue 1, pp. 81, (2022) (PubMed).
- : "A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets." in: Development (Cambridge, England), (2022) (PubMed).
- : "Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation." in: Nature methods, Vol. 19, Issue 11, pp. 1383-1392, (2022) (PubMed).
- : "Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag." in: Nature biotechnology, (2022) (PubMed).
