Chromatin profiling assays enable the mapping of protein-DNA interactions that influence chromatin modeling and gene expression.
Over the years, Chromatin ImmunoPrecipitation (ChIP) assays became the gold-standard for studying transcription factor–DNA binding interactions, nucleosome dynamics, and epigenetics (ex. histone post-translational modifications (PTMs)). This post aims at introducing the latest evolution of ChIP assays: CUTANA CUT&RUN and ChIC assays.
Over the last years, ChIP experts express the need for more sensitivity, faster protocols, and more reproducible results (and ideally starting from less starting cellular material). These expectations led to the emergence of new experimental antibody-based experimental approaches like Chromatin ImmunoCleavage (ChIC) assays, and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) methods.
CUTANA New ChIP assays
Based on ChIC / CUT&RUN technologies, CUTANA product line (developed by Epicypher and distributed by tebu-bio) is a novel ultra-sensitive genomic mapping assay that uses a proprietary immunotethering approach.
Similar to ChIC and CUT&RUN methods, it allows the fast mapping histone PTMs and chromatin interactors with high resolution.
CUTANA ChIP principle and advantages
This new approach leverages a factor specific antibody to tether a fusion of protein A, protein G and micrococcal nuclease (pAG-MNase) to genomic binding sites in intact cells, which is then activated by the addition of calcium to cleave DNA. The assay is performed in less than 4 days.
Compared to ChIP-seq assay, CUTANA assays have:
- lower sequencing requirements (Sequencing reads required: 3 million),
- improved signal-to-noise, reduced assay time (from cells to data in less than 4 days with < 1day of protocol time)
- less starting cellular material (100 cells required only as described by Dirks et al. in 2016 (Clin Epigenetics; 2016 (Nov 21); 8 :122).
Additionally, CUTANA assays save 5-10x in sequencing requirements and generate superior quality data compared to ChIP-seq using as little as 3 million sequencing reads.
Comparing data generated from standard ChIP-seq methods and sequencing depth (black) to CUTANA (blue). H3K27me3 and H3K4me3 ChIP-Seq data were sourced from ENCODE. Negative control data (yellow) was generated using a Rabbit IgG antibody in CUTANA. Each dataset of CUTANA was acquired using 0.5 million cells (source Epicypher).
Cells (or nuclei) are immobilized on lectin-coated magnetic beads, permeabilized, and incubated with an antibody to a chromatin target (e.g. histone PTM or chromatin / DNA binding protein). Next, pAG-MNase is added and activated via Ca2+. The clipped chromatin fragments diffuse out, followed by DNA purification and next-generation sequencing.
ChIP assays: pAG-MNase now available!
The pAG-MNase enzyme is the first reagent in the CUTANA product line and is available now from tebu-bio CUT&RUN & ChIC assays.
Recombinantly produced in E. coli, CUTANA pAG-MNase is a fusion of Proteins A and G to Micrococcal Nuclease and contains a C-terminal 6xHis epitope tag. 2 sizes are available:
- 50 Rxns – Cat. nr 15-1016
- 250 Rxns – Cat. nr 15-1116
Recently, a new protocol for CUT&RUN has been released (accessible here). Extensively optimized to ensure consistent yields, high reproducibility, and improved signal-to-noise across a variety of targets (histone PTMs, chromatin remodelers, and transcription factors), this comprehensive protocol includes notes and pictures throughout, providing clarity in sample handling at key steps and enabling a more thorough QC Analysis.
Extensively optimized to ensure consistent yields, high reproducibility, and improved signal-to-noise across a variety of targets (histone PTMs, chromatin remodelers, and transcription factors), this comprehensive protocol includes notes and pictures throughout, providing clarity in sample handling at key steps and enabling a more thorough QC Analysis.
NEW! SNAP-ChIP Certified, CUTANA Compatible Antibody
Histone H3K4me3 Antibody (cat. nr 13-0041 – 100 µg)
This rabbit recombinant Polyclonal (pool of multiple monoclonals (IgG)) is validated for ChIP, ChIP-Seq and CUT&RUN applications.
This antibody meets EpiCypher’s “SNAP-ChIP Certified” criteria for specificity and target enrichment in ChIP (<20% cross-reactivity to related histone post-translation modifications and >5% recovery of target input determined using SNAP-ChIP K-MetStat Panel spike-in controls; EpiCypher Catalog No. 19-1001).
Although its specificity in CUT&RUN has yet to be empirically determined in situ using spike-in controls, CUT&RUN data produced by this antibody shows a genome-wide enrichment pattern characteristic of H3K4me3 and is highly correlated with ChIP-seq.
Coming soon! CUTANA pAG-MNase assay kit for ChIC / CUT&RUN, as well as CUTANA pAG-Tn5 enzyme and assay kit for ChIC / CUT&TAG will be released. So stay tuned!
ChIP related products
- SNAP-ChIP certified antibodies
- Recombinant human mononucleosomes
- Chromatin remodeling enzymes and substrates
- Histone octamers and recombinant histones
CUTANA is a proprietary technology based on chromatin immuno-cleavage (ChIC) (US20070009937A1; Schmid et. al, Mol Cell 2004) and cleavage Under Targets and Release Using Nuclease (CUT&RUN) (patent pending; Skene and Henikoff, eLIFE 2017) methodology.
