Using Cleavage Under Targets and Tagmentation (CUT&Tag) Assay in Mouse Myoblast Research

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.