Using Cleavage Under Targets and Tagmentation (CUT&Tag) Assay in Mouse Myoblast Research
March 1st, 2024
Researchers new to the epigenetic field will find CUT&Tag a significantly easier alternative to ChIP assays. CUT&Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.
Our group's research focuses on how genome structure and transcription in adult skeletal muscle stem cells are regulated and maintained, particularly during cell phase changes like stem cell commitment, differentiation, and senescence. Adult skeletal muscle stem cells, comprising just 1%of mononucleated cells in muscle tissue, present a challenge for chromatin feature studies using Hi-C, ChIPs and ATAC-seq due to their scarcity in single cell suspensions obtained from muscle digestion. Chromatin immunoprecipitation, ChIP, has been a key tool for profiling genome features.
A simpler, more efficient contact technique developed by Dr.Steven Henikoff often surpasses ChIP, especially in studying epigenetics in rare cell populations. Its ease of use broadens accessibility for researchers, including non-epigenetic experts. To begin, add 30 microliters of Concanavalin A beads into a 1.5 milliliter centrifuge tube.
Pipette 300 microliters of binding buffer to the beads. Invert the tube multiple times to mix well. Then place the tube on a magnetic rack.
Once the solution is clarified, pipette out the supernatant. Pipette 300 microliters of binding buffer to the tube after removing it from the rack. Invert again to mix the contents of the tube.
Distribute the contents of the tube evenly into three tubes of an 8 PCR tube strip. Then place the tube strip on the magnetic rack until the solution is clarified. After removing the supernatant, pipette 10 microliters of binding buffer into the tubes and mix.
Place the prepared beads on ice until further experimentation. Next, trypsinize cultured mouse myoblasts off the plates. After centrifugation of the cells, use vacuum to remove supersede with vacuum.
Then re-suspend the pellet in PBS. Centrifuge the cells after performing a cell count of the suspension. Then, re-suspend the pellet in an adequate volume of wash buffer to generate a cell density of 700, 000 cells per milliliter.
Pipette 100 microliters of this cell suspension into each tube of the PCR tube strip and mix well. Then place the tube strip on a seesaw motion shaker for 10 minutes at room temperature to let the concanavalin A beads capture the cells. Clarify the contents of the tubes once more on a magnetic rack before pipetting out the supernatant.
Finally, observe the supernatant under a microscope to assess the efficacy of concanavalin A bead binding with the cells. To begin, pipette three microliters of H3K4me1 antibody into a tube containing 150 microliters of antibody buffer. Pipette the diluted antibody into each tube of an eight PCR tube strip, containing mouse myoblasts sequestered to concanavalin A beads.
Invert the tubes several times to mix the contents well, then place the tubes overnight on a seesaw motion shaker at four degrees Celsius. The next day, after clarifying the samples on a magnetic rack, pipette out the supernatant. Add 50 microliters of diluted secondary antibody into each sample tube after aspirating the supernatant.
Mix the sample tubes by inversion and incubate on a seesaw motion shaker for one hour at room temperature. Discard the supernatant of the clarified sample, then pipette 200 microliters of Dig wash buffer into each tube. Place the tubes back on the magnetic rack and pipette out the supernatant.
Next, add 100 microliters of 300-Dig buffer, containing pA/G-Tn5a into each tube. Once incubation is complete, place the tubes back on the magnetic rack. Then pipette out the clarified supernatant.
Now add 200 microliters of 300-Dig buffer into each tube. Place the sample tubes on a shaker for three minutes at room temperature. After the last wash, thoroughly pipette out the supernatant from each magnetically clarified tube.
Then add 50 microliters of tagmentation buffer. Finally, incubate the tubes in a PCR machine at 37 degrees Celsius for one hour without using the lid heating function of the instrument. To begin, add 150 microliters of tagmentation buffer into the sample tubes containing the tagmented mouse myoblast samples.
Pipette EDTA, SDS, and proteinase K into each tube. Vortex the contents of the tubes to mix well, then incubate the tubes at 55 degrees Celsius for two hours in a PCR machine. Next, pipet each sample into 1.5 milliliter centrifuge tubes containing 200 microliters of phenol chloroform mixture.
Vortex the contents of the tubes to mix them. Once centrifugation is complete, transfer the top layer into a new 1.5 milliliter centrifuge tube. Then add 200 microliters of chloroform into the new tubes before vortexing and centrifuging again.
Aspirate the top layer into a new tube. Now add 550 microliters of 100%ethanol into each tube and mix well by inverting. Then place the tubes at minus 80 degrees Celsius for one hour to induce DNA precipitation.
Centrifuge the solution at maximum speed for 15 minutes to obtain a DNA pellet. After pouring out the supinate, wash the pellet with 75%ethanol before centrifuging again for five minutes. Then re-suspend the pellet in 21 microliters of double distilled water.
Set up the PCR to be used for the library preparation. Use a different N7 XX Illumina primer for each sample and a universal N5 XX primer. Input the PCR cycle number into the instrument to run the PCR.
After amplification, electrophoresis three microliters of the libraries on aeros gels. The cut and tag libraries mostly contained tagged mono-nucleosomes of about 300 base pairs. QPCR showed that the H3K4me1 mark was highly enriched at the enhancer regions of the Myod1 gene.
Overview
This article discusses the advantages of the CUT&Tag technique over traditional ChIP assays for studying epigenetics in rare cell populations, specifically in adult skeletal muscle stem cells. The protocol focuses on performing H3K4me1 CUT&Tag assays on mouse myoblasts isolated from hindlimb muscles.
Key Study Components
Area of Science
- Epigenetics
- Cell Biology
- Muscle Stem Cell Research
Background
- Adult skeletal muscle stem cells are scarce, comprising only 1% of mononucleated cells in muscle tissue.
- Traditional methods like ChIP and ATAC-seq face challenges due to the low availability of these cells.
- CUT&Tag is a simpler and more efficient technique for profiling chromatin features.
- This method enhances accessibility for researchers, including those not specialized in epigenetics.
Purpose of Study
- To demonstrate the effectiveness of CUT&Tag in studying epigenetic modifications in rare cell populations.
- To provide a detailed protocol for conducting H3K4me1 CUT&Tag assays.
- To facilitate research in the field of epigenetics for a broader range of scientists.
Methods Used
- Isolation of mouse myoblasts from hindlimb muscles.
- Preparation of Concanavalin A beads for the CUT&Tag assay.
- Application of the CUT&Tag protocol to analyze H3K4me1 modifications.
- Data collection and analysis of epigenetic features.
Main Results
- CUT&Tag provides high-quality data from very few cells.
- The technique successfully profiles epigenetic modifications in muscle stem cells.
- Results demonstrate the advantages of CUT&Tag over traditional methods.
- Findings contribute to a better understanding of genome regulation in muscle stem cells.
Conclusions
- CUT&Tag is a valuable tool for researchers studying epigenetics in rare cell populations.
- The protocol enhances the ability to generate meaningful data from limited samples.
- This study supports the broader application of CUT&Tag in various biological research fields.
Frequently Asked Questions
Executive Industry Relevance
Profiling chromatin features in rare cell populations is a critical challenge in early discovery and target validation, especially for muscle stem cell research. The CUT&Tag assay enables high-sensitivity mapping of chromatin binding factors and histone marks from minimal cell numbers, supporting predictive confidence in epigenetic target identification. This capability streamlines portfolio triage and de-risks early-stage mechanistic hypotheses in muscle and stem cell biology.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of chromatin state and transcriptional regulation in rare cell populations.
- Supports functional target validation by mapping histone marks and chromatin factors at specific genomic loci.
- Improves predictive confidence for epigenetic targets by reducing sample bias and background noise.
- Facilitates portfolio triage by providing robust data from limited biological material.
Screening & Assay Development
- Prepares validated chromatin profiling systems for downstream compound screening workflows.
- Delivers reproducible, quantitative readouts of histone modifications and chromatin factor binding.
- Enables assay standardization and scalability due to low cell input requirements.
- Supports reliable evaluation of epigenetic modulators in screening campaigns.
Translational & Preclinical Research
- Aligns chromatin profiling outputs with disease-relevant muscle stem cell models.
- Maintains translational continuity from discovery through preclinical validation of epigenetic mechanisms.
- De-risks advancement decisions by providing unbiased, high-resolution chromatin data.
- Supports biomarker alignment for muscle regeneration and differentiation studies.
Pipeline & Workflow Integration
The CUT&Tag assay integrates into the discovery continuum from early target validation through preclinical research, particularly where cell scarcity limits traditional ChIP-based approaches.
- Discovery Biology: Enables hypothesis testing and pathway clarification in rare muscle stem cell populations.
- Screening: Provides assay-ready, reproducible chromatin profiling for compound evaluation.
- Analytics: Generates quantitative, low-background sequencing data for robust condition comparison.
- Translational Research: Connects chromatin state mapping to disease-relevant muscle models and biomarker strategies.
- Enterprise Reuse: Offers a scalable, broadly applicable platform for chromatin profiling across diverse cell types.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in epigenetic target validation.
- Operational Value: Streamlines workflows with fewer steps, lower cell input, and higher sensitivity than ChIP.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by delivering robust data from scarce samples.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of epigenetic programs.
Implementation Considerations
- Requires expertise in chromatin biology and next-generation sequencing analysis.
- Needs access to magnetic bead handling, PCR, and sequencing infrastructure.
- Demands cross-team standardization for reproducibility and data comparability.
- Adaptable to various cell types if immobilization with Concanavalin-A beads is feasible.
- Limited by the need for high-quality antibodies and careful sample handling to minimize bias.
Biopharma FAQs
Chapters in this video
0:00
Introduction
1:15
Sequestration of Mouse Myoblasts onto Activated Concanavalin‐A Beads
3:08
Incubation of Mouse Myoblast‐Sequestered Concanavalin‐A Beads with Antibodies for CUT&Tag Assay
4:59
Genomic Purification and Library Preparation of Tagmented Mouse Myoblast DNA for Epigenetic Profiling
