This work was supported by the Strategic Priority Research Program of the Chinese Academy of Science (XDA16020400 to PH); the National Natural Science Foundation of China (32170804 to PH).

The methods presented in this manuscript are all approved by the Institutional Animal Care and Use Committee of Guangzhou Laboratory. Mice used to generate this manuscript&#39;s representative results were housed and maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Guangzhou Laboratory.
1. Myoblast isolation from mouse hindlimb muscles (Example of using 1 mouse)
<ol>
	<li>Dilute glacial acetic acid with ddH2O to 0.02 M and autoclave it.</li>
	<li>Dilute collagen (from rat tail) to 0.1 mg/mL with 0.02 M acetic acid.</li>
	<li>Add this collagen solution into culture dishes to coat the dish bottom in a 37&#160;&#176;C cell incubator for overnight.</li>
	<li>Before use, remove the collagen solution and wash the dish twice with 1x PBS. Collagen solution can be reused for 8-10 times.</li>
	<li>Sacrifice an 8-12-week-old mouse. (Old mice have poor yields of MuSCs and myoblasts.)</li>
	<li>Isolate and place all hindlimb muscles from the mouse into a 10-cm dish.</li>
	<li>Use 1x PBS (with penicillin/streptomycin) to rinse the muscles 4 times. After each rinse, always transfer the muscles into a new plate.</li>
	<li>After the final rinse, remove PBS and mince up the muscles with scissors.</li>
	<li>Add 6 mL of 1x PBS containing dispase II (1.1 U/mL) and collagenase II (830 U/mL) to digest the minced muscles, and the digestion should last for ~1.5 h.</li>
	<li>To perform digestion, transfer the minced muscles into a tube in a 37 &#176;C water bath or directly leave the minced muscles within the dish and place it in a 37 &#176;C CO2 cell incubator.</li>
	<li>If using a cell incubator to digest, rock the dish every 15-30 min to mix the digestion well. 
	NOTE: Dispase II should be first made as 11 U/mL stock with DMEM media (no serum), and collagenase II should be made as 10,000 U/mL stock with 1x PBS.</li>
	<li>When the digestion is complete, use a 10-mL plastic pipet to flush the digested tissues up and down several times to homogenize the digestion mixture thoroughly.</li>
	<li>Add 10 mL of MuSC/Myoblast growth media to slow down the digestion. Details on the preparation of MuSC/Myoblast growth media can be found in Section 2.</li>
	<li>Run the mixture through a 70-&#181;m strainer. Use 1x PBS to rinse the digestion plate and run this PBS through the strainer as well, in order to collect all the cells in the digestion plate.</li>
	<li>Centrifuge at 350 x g for 10 min and discard the supernatant with vacuum.</li>
	<li>Resuspend the cell pellet with 20 mL of 1x PBS and run the cells through a 40-&#181;m strainer.</li>
	<li>Centrifuge at 350 x g for 5 min and remove the supernatant with vacuum.</li>
	<li>Use 20 mL of MuSC/Myoblast growth media to resuspend the cells and seed the cells in a 10-cm normal plastic plate. MuSCs cannot attach to the plate bottom during this time and will stay floating mostly in the media. 
	NOTE: This is the first pre-plating on plastic plates.</li>
	<li>After 1.5 h, briefly rock the plate and transfer the liquid into two collagen-coated 10-cm dishes, each dish containing 10 mL of culture. Now, the MuSCs will attach and grow on the collagen-coated plates. Change the media after 2 days. Collagen-coated plates are prepared as in steps 1-4 of this section.</li>
	<li>After another day, passage the cells: Remove the media, wash the cells once with 1x PBS, and trypsinize the cells.</li>
	<li>Use MuSC/Myoblast growth media to flush the cells off the plates, move the cells onto a new plastic plate, and incubate in the cell incubator for 40 min. 
	NOTE: This is the second pre-plating on plastic plates.</li>
	<li>Pour the culture into a new plastic plate and incubate in the cell incubator for another 20 min. 
	NOTE: This is the third pre-plating on plastic plates.</li>
	<li>Then split the supernatant in the plate into 8 collagen-coated 10-cm dishes to subculture the cells. The passage ratio here is 2: 8 (1: 4). After three times of pre-plating described above, the myoblasts can reach over 90% pure. Therefore, after this point, there is no need to pre-plate when further passaging the myoblasts.</li>
</ol>
2. Preparation of MuSC/Myoblast growth media (Example of preparing from 5 mice)
<ol>
	<li>Sacrifice 5 young and healthy wild-type mice, isolate the spleens, and rinse the spleens once with 1x PBS (with penicillin/streptomycin).</li>
	<li>Use scissors to remove the dark parts/spots on the spleens, if any. Discard the spleen if most of it has turned black.</li>
	<li>Place all 5 spleens into a 40-&#181;m strainer on the top of a 50 mL tube.</li>
	<li>Open a sterile 1 mL syringe package, pull out the plunger, and discard the barrel.</li>
	<li>In the 40-&#181;m strainer, hold the rubber stopper and use the thumb rest of the plunger to grind and disassociate the spleen tissues into single cells. Be careful not to touch and contaminate the thumb rest of the plunger when tearing open the syringe package.</li>
	<li>While grinding the spleens, occasionally use 1x PBS to wash off the dissociated spleen cells into the 50 mL tube underneath the strainer.</li>
	<li>After all the spleen cells have been flushed into the 50 mL tube through the strainer, centrifuge the tube at 500 x g for 10 min.</li>
	<li>Use 1 mL of 1x Red blood cell lysis buffer to resuspend the pellet. Lyse the red blood cells for ~1 min. 
	NOTE: 10x Red blood cell lysis buffer contains 155 mM NH4Cl, 10 mM NaHCO3, and 1 mM EDTA disodium salt in ddH2O. Filter to sterilize, and do not autoclave. Dilute with ddH2O to 1x before use.</li>
	<li>Fill up the 50 mL tube with 1x PBS to cease the lysing process of red blood cells.</li>
	<li>Strain the cell suspension again with a 40-&#181;m strainer, and then centrifuge at 500 x g for 10 min.</li>
	<li>Resuspend the cells with 2 mL of RPMI-1640 full media (RPMI-1640 media with 10% FBS and penicillin/streptomycin) with very gentle pipetting.</li>
	<li>Add more RPMI-1640 full media into the tube and mix well. Distribute the cell suspension into ten T75 culture flasks. The final culture volume in each T75 culture flask should be 20 mL.</li>
	<li>Add 10 &#181;L of Concanavalin-A stock into each T75 culture flask and mix well. Concanavalin-A activates the proliferation of T-cells. 
	NOTE: Concanavalin-A stock is 4 mg/mL and prepared by dissolving Concanavalin-A in 1x PBS. The Concanavalin-A itself is used here, and do not confuse it with the Concanavalin-A beads used in CUT&#38;Tag assays later on.</li>
	<li>After 48 h, supplement each T75 flask with another 20 mL of RPMI-1640 full media so the total volume will add up to 40 mL.</li>
	<li>Another 24 h later, collect all the culture in each T75 flask and centrifuge at 600 x g for 3 min.</li>
	<li>The supernatant is T-cell conditional media and can be stored at -20 &#176;C to -80 &#176;C for up to 2 months. Discard the spleen cell pellets. Before use, thaw the T-cell conditional media and further sterilize the media using a 0.22 &#181;m filter.</li>
	<li>To make Ham&#39;s F10 full media, add 100 mL of FBS, 300 &#181;L of bFGF stock, and 6 mL of penicillin/streptomycin into 500 mL of Ham&#39;s F10 media. 
	NOTE: bFGF stock is 5 &#181;g/mL and prepared by dissolving bFGF in Ham&#39;s F10 media.</li>
	<li>Mix the Ham&#39;s F10 full media with filtered T-cell conditional media described above at 1:1 ratio to make MuSC/Myoblast growth media.</li>
</ol>
3. CUT&#38;Tag with myoblasts (Example of 3 CUT&#38;Tag reactions)
<ol>
	<li>Preparation of key reagents
	<ol>
		<li>Purify the pA/G-Tn5 transposase in-house from bacterial culture using published method14. This enzyme only reaches its functional form till it is mounted with library-making DNA adaptors. Most importantly, pA/G-Tn5 is commercially available from many sources.</li>
		<li>The oligos to make adaptors are shown in Table 1. Specifically, anneal Oligo-Rev with Oligo-A and Oligo-B, respectively, to make double-stranded Adaptor-A and Adaptor-B accordingly. The details for annealing to make the adaptors can be found elsewhere14.</li>
		<li>Incubate Adaptor-A and Adaptor-B with pA/G-Tn5 transposase at room temperature (RT) for 2 h. Then, this transposase will take its functional form. In the following sections of this manuscript, the functional form of pA/G-Tn5 will be termed pA/G-Tn5a. 
		NOTE: If the pA/G-Tn5 is purchased instead of in-house made, make sure to clarify whether the bought item is pA/G-Tn5 mounted with adaptors (in other words, ready to use) or it is just the pA/G-Tn5 enzyme itself. If it is just the enzyme, prepare the two adaptors described above and mount them onto the pA/G-Tn5 before finally using this enzyme for CUT&#38;Tag.</li>
		<li>For the recipes of the Binding buffer, Wash buffer, Dig-wash buffer, 300-Dig buffer, and Tagmentation buffer, readers can also refer to Kaya-Okur et al.1.</li>
		<li>Prepare the Binding buffer, which contains 20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2,&#160;and 1 mM MnCl2.</li>
		<li>Prepare the Wash buffer, which contains 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and 1x Protease inhibitor cocktail.</li>
		<li>Make digitonin as a concentrated stock in DMSO, and then prepare Dig-wash buffer by adding digitonin into Wash buffer to a concentration of 0.5 mg/mL.</li>
		<li>Prepare Antibody buffer by adding EDTA to a concentration of 2 mM and BSA to a concentration of 0.1%, into the Dig-wash buffer.</li>
		<li>Prepare 300-Dig buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Protease inhibitor cocktail, and 0.1-0.5 mg/mL digitonin.</li>
		<li>Prepare the Tagmentation buffer by supplementing the 300-Dig buffer with 10 mM MgCl2. MgCl2 can activate the enzymatic activity of pA/G-Tn5a. 
		NOTE: Information for other materials is provided in the Table of Materials file.</li>
	</ol>
	</li>
	<li>Concanavalin-A bead activation
	<ol>
		<li>Mix 30 &#181;L of Concanavalin-A beads (10 &#181;L for each reaction) into 300 &#181;L of Binding buffer in a 1.5 mL centrifuge tube. Invert several times to mix well. Put the tube onto a magnetic rack. After the liquid is clear, remove the supernatant.</li>
		<li>Remove the tube from the magnetic rack, add 300 &#181;L of Binding buffer in the tube, mix well, and evenly split into 3 tubes of an 8-PCR tube stripe. Designate each tube with one CUT&#38;Tag reaction.</li>
		<li>Put the 8-PCR tube stripe on a magnetic rack and wait till the liquid is clear.</li>
		<li>Aspirate the supernatant and remove the tubes from the magnetic rack. Add 10 &#181;L of Binding buffer into each tube and mix well. Place the prepared beads on ice or at 4&#160;&#176;C till the cells are ready to proceed.</li>
	</ol>
	</li>
	<li>Binding cells onto Concanavalin-A beads
	<ol>
		<li>Trypsinize cultured mouse myoblasts off the plates, wash the cells once with RT 1x PBS and eventually resuspend the cells in Wash buffer at RT. Avoid over-trypsinization, as this can compromise the affinity of the cell membrane to Concanavalin-A beads.</li>
		<li>Adjust the cell density to around 7 x 105 cells/mL in Wash buffer such that 100 &#181;L of cell suspension contains roughly 70,000 cells, sufficient for one CUT&#38;Tag reaction.</li>
		<li>Pipet 100 &#181;L of the cell suspension into each tube of the 8-PCR tube stripe, which already contains 10 &#181;L of prepared Concanavalin-A beads. Mix well and incubate on a &#34;seesaw motion&#34; shaker for 10 min at RT.</li>
		<li>Put the 8-PCR tube stripe on a magnetic rack. Wait till it clears, and remove the supernatant.</li>
		<li>To assess whether the Concanavalin-A beads have efficiently sequestered the cells, check the supernatant under a microscope to see how many cells are left in the supernatant. After the Concanavalin-A bead binding step, very few or no cells should be observed in the supernatant.</li>
	</ol>
	</li>
	<li>Incubating the cells with a primary antibody
	<ol>
		<li>For each sample, dilute 1 &#181;L of H3K4me1 antibody into 50 &#181;L of Antibody buffer and add the diluted antibody into each sample tube. Mix the beads well with the diluted antibody by inverting the tubes several times. Put the samples on the &#34;seesaw motion&#34; shaker to incubate at 4&#160;&#176;C for overnight. 
		NOTE: As mentioned by Dr. Steven Henikoff1, it was not necessary to use an IgG negative control in all CUT&#38;Tag assays. Compared to ChIPs, CUT&#38;Tag assays come with much lower or no background signal. Therefore, using IgG to perform a &#34;negative control&#34; CUT&#38;Tag assay does not introduce any useful information. In contrast, using a CUT&#38;Tag-validated antibody to perform a positive control is important to assess whether the CUT&#38;Tag assay has worked.</li>
	</ol>
	</li>
	<li>Incubating the cells with a secondary antibody
	<ol>
		<li>The next day morning, put the samples on the magnetic rack and wait till the samples clear.</li>
		<li>Remove the supernatant, which is the primary antibody. There is no need to wash the beads; Directly add 50 &#181;L of diluted (1:100) secondary antibody into each sample tube. 
		NOTE: Secondary antibody is diluted with Dig-wash buffer at a 1:100 ratio. To allow more efficient pA/G-Tn5a recognition, secondary antibodies should not be conjugated with any molecules or groups such as HRP, biotin, or fluorophores.</li>
		<li>Mix well and incubate on the &#34;seesaw motion&#34; shaker at RT for 1 h.</li>
		<li>Put the samples back on the magnetic rack, wait till the samples clear, and discard the supernatant.</li>
		<li>Add 200 &#181;L of Dig-wash buffer into each tube and invert several times to wash off the excessive antibodies.</li>
		<li>Put it back on the magnetic rack, wait till it clears, and remove the supernatant. Repeat this washing twice more to completely remove excessive unbound antibodies.</li>
	</ol>
	</li>
	<li>Incubating the cells with pA/G-Tn5a
	<ol>
		<li>After the last wash, put the samples on the magnetic rack, wait till the samples clear, and remove all the liquid in the tube. For each sample tube, add 100 &#181;L of 300-Dig buffer containing pA/G-Tn5a at 0.04 &#181;M. Mix well and incubate on the seesaw motion shaker for 1 h at RT.</li>
		<li>Put on the magnetic rack. Wait till the samples clear and remove the supernatant.</li>
		<li>Add 200 &#181;L of 300-Dig buffer into each sample, mix well, and place on the shaker to wash the sample for 3 min at RT.</li>
		<li>Put on the magnetic rack. Wait till the samples clear and remove the supernatant. Repeat this washing twice more to remove excessive pA/G-Tn5a and lower the background.</li>
	</ol>
	</li>
	<li>Cleavage and tagmentation of the genomes
	<ol>
		<li>After the last wash, put the samples on the magnetic rack and thoroughly pipet the liquid off the tube. For each sample, add 50 &#181;L of Tagmentation buffer. Mix well and incubate in a PCR machine at 37&#160;&#176;C for 1 h. Do not use the lid-heating function of the PCR machine. 
		NOTE: Optionally, due to the material wearing during the following genomic DNA purification step, some spike-in DNAs can be added into the Tagmentation buffer so that the spike-in will show up in the final sequencing results and can be used to normalize the data. The method for preparing and using the spike-in DNA can be found elsewhere14.</li>
	</ol>
	</li>
	<li>Total genomic DNA purification
	<ol>
		<li>After tagmentation, add another 150 &#181;L of Tagmentation buffer for each sample. The total volume is now 200 &#181;L.</li>
		<li>Then, for each sample, add 10 &#181;L of 0.5 M EDTA, 3 &#181;L of 10% SDS, and 2.5 &#181;L of 20 mg/mL Proteinase K. Vortex to mix well and incubate at 55&#176;C for 2 h.</li>
		<li>Pipet each sample into 200 &#181;L of phenol/chloroform in a 1.5 mL centrifuge tube and vortex for 10 s. Centrifuge at 18,000 x g for 5 min. Transfer the top layer into a new 1.5 mL centrifuge tube.</li>
		<li>Add 200 &#181;L of chloroform into each tube and vortex for 10 s. Centrifuge at 18,000 x g for 5 min.</li>
		<li>Aspirate the top layer into a new 1.5 mL centrifuge tube. Then, for each sample, add 550 &#181;L of 100% ethanol to precipitate the DNA at -80&#160;&#176;C for 1 h. To better visualize the DNA pellet, add 1 &#181;L of 20 mg/mL glycogen.</li>
		<li>Centrifuge at max speed (&#62;18,000 x g) for 15 min to pellet the DNA.</li>
		<li>Carefully pour off the supernatant, wash the glycogen/DNA pellet with 75% ethanol once, and centrifuge again at max speed for 5 min.</li>
		<li>Remove the 75% ethanol and resuspend the glycogen/DNA pellet with 21 &#181;L of ddH2O.</li>
	</ol>
	</li>
	<li>Library preparation and sequencing
	<ol>
		<li>Set up the library-preparing PCR. To index the samples to be sequenced together, use a different N7XX Illumina primer for each sample and a universal N5XX Illumina primer. If indexing more than 12 samples, then different N5XX Illumina primers will be required as well. Illumina provides twelve N7XX primers and eight N5XX primers, so the total number of N7XX with N5XX combinations is 12 x 8 = 96. The PCR reaction setup is shown in Table 2.</li>
		<li>Roughly determine the PCR cycle number in library-preparing PCR by the cell number used in the CUT&#38;Tag assay. Normally, for a CUT&#38;Tag assay using 50,000-100,000 cells, 9-12 cycles are quite sufficient, and cell numbers between 10,000-50,000 might require up to 14 cycles. The PCR program is displayed in Table 3. 
		NOTE: For most CUT&#38;Tag assays, if the antibody has functioned properly, then a PCR cycle number between 9-14 should always generate a useful library for sequencing. A good library is normally 10-50 ng/&#181;L (determined by Qubit assay) in concentration and 20-30 &#181;L in volume. On the contrary, if the antibody did not work efficiently and specifically, then merely increasing the cycle number cannot save the experiments. Excessive cycle numbers do not benefit the results at all and instead introduce more PCR duplicates, which will later complicate the data analysis.</li>
		<li>Use some commercially available DNA purification/size-selection beads to purify the library DNA and select the DNA of desirable sizes.</li>
		<li>Dilute a small aliquot of the purified library with water and use it for qPCR with locus-specific primers to check enrichments as in ChIP-qPCR experiments. Before sequencing the library, this can help determine whether the CUT&#38;Tag has worked.</li>
		<li>Perform sequencing and data analysis as per the manufacturer&#39;s instructions. 
		NOTE: CUT&#38;Tag kits are commercially available from a handful of companies, and one example can be found in the&#160;Table of Materials file.</li>
	</ol>
	</li>
</ol>

Profiling Genomic Histone Marks in Mouse Myoblasts Using CUT&#38;Tag

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+transcriptional+activation+of+MyoD+mediated+by+replication-independent+histone+deposition.">Myogenic transcriptional activation of MyoD mediated by replication-independent histone deposition.</a> Proc Natl Acad Sci U S A. 108 (1), 85-90 (2011).</li><li>Kaya-Okur, H. S., Janssens, D. H., Henikoff, J. G., Ahmad, K., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+low-cost+chromatin+profiling+with+CUT&amp;Tag.">Efficient low-cost chromatin profiling with CUT&amp;Tag.</a> Nat Protoc. 15 (10), 3264-3283 (2020).</li></ol>

The authors have nothing to disclose.

The specific cell number required in a certain CUT&#38;Tag reaction completely relies on the enrichment of the histone marks/variants or chromatin-binding proteins that are to be tested. Normally for very enriched histone marks such as H3K4me1, H3K4me3, and H3K27ac etc., 25,000-50,000 myoblasts are quite sufficient for one CUT&#38;Tag reaction. However, some rare chromatin-binding proteins might require up to 250,000 cells. The cell number used in CUT&#38;Tag assays is critical, which, if not handled well normally causes...

CUT&#38;Tag assay was invented to compensate for some overt flaws of ChIPs1. The two major disadvantages of ChIPs are 1) the bias introduced when fragmenting chromatin and 2) the incompetence to work with low cell numbers. X-ChIP assays rely on either sonication or MNase digestion to get chromatin fragments, whereas N-ChIP mostly uses MNase digestion to get nucleosomes. Sonication shows a bias towards relaxed chromatin locations such as promoter regions2, and apparently, MNase digestion also works more efficiently on relaxed chromatin fibers. Moreover, some reported that MNase digestion also shows a DNA sequence-dependent bias3. Therefore, at the input preparation step of ChIP assays, it is impossible to get chromatin fragments from all kinds of genomic locations in a perfectly random manner. Moreover, ChIP assays normally generate higher background signals compared to CUT&#38;Tag and require over 10 folds more reads than CUT&#38;Tag to accentuate where the peaks are1,4,5. This explains why ChIP experiments have to start with tremendously more cells than CUT&#38;Tag. This is not a problem when studying cell lines as they can be repetitively passaged to achieve a very high cell number. However, the ChIP assay is definitely not a strong epigenetic tool to study rare or precious primary cell populations, although primary cells obviously hold more practical and medical implications.
While the long and complicated ChIP procedure discourages some researchers from learning or using this technique, people are more comfortable with easier assays such as immunocytochemistry (ICC) or immunofluorescence (IF). A CUT&#38;Tag assay essentially resembles the process of ICC and IF experiments but only takes place in a test tube. CUT&#38;Tag does not need fragmented chromatin to start with, and instead, the genome must be intact for antibody binding1. On the first day of a ChIP experiment, researchers normally spend up to 4 h preparing the fragmented chromatins from nuclei with sonication or MNase digestion before the short chromatin pieces can be mixed with antibody-beads4,5. In remarkable contrast, the first-day workload of a CUT&#38;Tag procedure is to just immobilize the cells to Concanavalin-A beads and then to add the primary antibody onto the cell-beads. This only requires ~40 min1.
It is worth mentioning that Cleavage Under Targets and Release Using Nuclease (CUT&#38;RUN) is an important alternative to CUT&#38;Tag. CUT&#38;RUN was established based on a similar working mechanism as CUT&#38;Tag. In CUT&#38;Tag, the antibodies guide the pA/G-Tn5 transposase to all the locations where the enzyme will each cut out a piece of chromatin and meanwhile tag it with library-making adaptors, while in CUT&#38;RUN, the role of pA/G-Tn5 is played by the pA/G-MNase which only performs the cutting part of the job6. Therefore, compared to CUT&#38;Tag, CUT&#38;RUN requires an additional step which is to glue the library-making adaptors onto the pA/G-MNase-fragmented DNA pieces7,8. Due to the high similarities between CUT&#38;Tag and CUT&#38;RUN, researchers familiar with CUT&#38;Tag will easily adapt themselves to performing CUT&#38;RUN proficiently. However, it should be noted that some minor differences exist between CUT&#38;Tag and CUT&#38;RUN. CUT&#38;RUN protocols normally use the physical concentration of salt (~150 mM) in the washing steps, while in CUT&#38;Tag, the 300-Dig wash buffer is high in salt. Therefore, CUT&#38;Tag is good at controlling the background when profiling histone marks/variants or transcription factors, as these proteins directly and strongly bind DNA1. CUT&#38;Tag may run into problems when profiling chromatin-associated factors that do not directly bind DNA and show weak affinity to the chromatin. High salt washing steps in CUT&#38;Tag may strip off chromatin-associated factors and cause no signals in the final output. Although there are successful cases where CUT&#38;Tag can be used to profile some non-histone/non-transcription factor proteins9, we still recommend CUT&#38;RUN over CUT&#38;Tag to profile weakly-bound chromatin-associated proteins.
After mammals reach adulthood, their skeletal muscle tissues still contain muscle stem cells. During muscle injury, these stem cells can be activated and undergo cell number expansion and differentiation to regenerate damaged muscle fibers10. These stem cells are known as Muscle stem/satellite cells (MuSCs). After MuSCs are isolated from animals or once activated by muscle injury, they start proliferating and become myoblasts.
To obtain MuSCs from mouse skeletal muscle digest, MuSC surface markers such as Vcam1 (Cd106), Cd34, and &#945;7-integrin (Itga7) are often used individually or in combinations to enrich MuSCs during fluorescence-activated cell sorting (FACS)11. It has been shown that Cd31-/Cd45-/Sca1-/Vcam1+ is probably the best marker combination to get &#62;95% pure MuSCs12. FACS can isolate pure MuSCs right after fresh muscles are digested. However, if the experimental design does not require pure MuSCs right at their isolation, pre-plating is more cost-effective than FACS to obtain &#62;90% pure myoblasts (MuSC progeny).
MuSCs freshly isolated from mice do not proliferate efficiently in Ham&#39;s F10 media supplemented with fetal bovine serum (FBS). To better expand the cell number of MuSCs and get sufficient myoblasts, bovine growth serum (BGS) should be used instead of FBS. However, if BGS is not available, Ham&#39;s F10 full media (containing about 20% FBS) can be mixed with an equal volume of T-cell conditional media to dramatically promote myoblast expansion13. Therefore, this protocol will also describe the preparation of T-cell media conditioned MuSC media.
Most importantly, this protocol provides a complete example of performing H3K4me1 CUT&#38;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles. Please note that this protocol also applies to other cell types and histone marks and histone variants, and readers only need to optimize the cell numbers or antibody amounts for their cases based on the enrichment of the specific histone marks or variants they study.
In order to be used in CUT&#38;Tag or CUT&#38;RUN, the Tn5 or MNase needs to be fused with protein A or protein G to make pA-Tn5, pG-Tn5, pA-MNase, or pG-MNase. Apparently, both protein A and protein G can be fused onto these enzymes at the same time to generate pA/G-Tn5 or pA/G-MNase. Protein A and protein G show differential affinities to IgGs from different species. Therefore, fusing protein A and protein G altogether onto the enzyme can overcome this problem and make the enzyme compatible with antibodies from multiple species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin-A</td>
			<td>Sigma-Aldrich</td>
			<td>C5275</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin-A beads</td>
			<td>Bangs Laboratories</td>
			<td>BP531</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>300410</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Thermo Fisher Scientific</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me1 antibody&#160;</td>
			<td>abcam</td>
			<td>ab8895</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F10 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11550043</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyperactive Universal CUT&#38;Tag Assay Kit for Illumina&#160;</td>
			<td>Vazyme</td>
			<td>TD903</td>
			<td>This kit has been tested by us to function well</td>
		</tr>
		<tr>
			<td>Magnetic rack for 1.5 mL EP tubes</td>
			<td>Qualityard</td>
			<td>QYM06</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic rack for 8-PCR tube stripes</td>
			<td>Anosun Magnetic</td>
			<td>CLJ16/21-021</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext High-Fidelity 2x PCR Master Mix</td>
			<td>NEB</td>
			<td>M0541L</td>
			<td>For library-making PCR reaction</td>
		</tr>
		<tr>
			<td>pA-Tn5</td>
			<td>Vazyme</td>
			<td>S603-01</td>
			<td>Needs to be mounted with adaptors before use</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Beyotime</td>
			<td>ST535-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody (Guinea Pig anti-rabbit IgG)</td>
			<td>Antibodies-online</td>
			<td>ABIN101961</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr>
			<td>TruePrep Index Kit V2 for Illumina&#160;</td>
			<td>Vazyme</td>
			<td>TD202</td>
			<td>This kit provide Illumina N7XX and N5XX primers&#160;</td>
		</tr>
		<tr>
			<td>VAHTS DNA Clean Beads&#160;</td>
			<td>Vazyme</td>
			<td>N411</td>
			<td>Can substitute Ampure XP beads. Can purify CUT&#38;Tag libraries and select DNA fragments by size</td>
		</tr>
	</tbody>
</table>

using cleavage under targets and tagmentation (cut&#38;tag) assay in mouse myoblast research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&amp;Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&amp;Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&amp;Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&amp;Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Before binding cells to Concanavalin-A beads, check the cell suspension under the microscope. Accordingly, after incubating the cells with Concanavalin-A beads, put the sample tubes on the magnetic rack, and the supernatant should be again observed using a microscope. This is to assess how efficiently the cells have been captured by Concanavalin-A beads. Wash buffer containing 7 x 105 cells/mL should look like Figure 1A under the microscope. In contrast, Figur...

Watch this Scientific Journal Video about Author Spotlight: Navigating Challenges and Innovations in Muscle Stem Cell Studies at JoVE.com

Author Spotlight: Navigating Challenges and Innovations in Muscle Stem Cell Studies

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;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&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile ...

using-cleavage-under-targets-tagmentation-cut-tag-assay-mouse

Research

JoVE Journal

Developmental Biology

526 Views.  Guangzhou Laboratory. 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.

Video: Author Spotlight: Navigating Challenges and Innovations in Muscle Stem Cell Studies

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...