incubation of mouse myoblast-sequestered concanavalin-a beads with antibodies for cut&#38;tag assay

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

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.

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

在小鼠成肌细胞研究中使用靶标和标记下的切割（CUT&#38;Tag）测定

小鼠成肌细胞隔离的刀豆球蛋白-A 珠与抗体一起孵育用于 CUT&#38;Tag 测定

首先，将 3 微升 H3K4me1 抗体移液到含有 150 微升抗体缓冲液的试管中。将稀释的抗体移液到包含小鼠成肌细胞的八个PCR管条的每管中，这些细胞被隔离到刀豆球蛋白A珠上。将试管倒置数次，以充分混合内容物。然后将试管放在 4 摄氏度的跷跷板运动振荡器上过夜。第二天，在磁性架上澄清样品后，用移液液出上清液。吸出上清液后，将 50 μL 稀释的二抗加入每个样品管中。通过倒置混合样品管，并在室温下在跷跷板运动振荡器上孵育一小时。丢弃澄清样品的上清液。然后将 200 微升 DIG 洗涤缓冲液移液到每个试管中。将试管放回磁性架上，然后用吸液吸出上清液。接下来，将 100 微升含有 pA/G-Tn5a 的 300 DIG 缓冲液加入每个试管中。孵育完成后，将试管放回磁性架上。然后用移液管取出澄清的上清液。现在向每个试管中加入 200 微升 300 DIG 缓冲液。将样品管在室温下放在振荡器上三分钟。最后一次洗涤后，从每个磁性澄清管中彻底吸取上清液。然后加入 50 微升标记缓冲液。最后，将试管在 37 摄氏度的 PCR 机中孵育一小时，而不使用仪器的盖子加热功能。

Research

JoVE Journal

Developmental Biology

Incubation of Mouse Myoblast-Sequestered Concanavalin-A Beads with Antibodies for CUT&#38;Tag Assay

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 ...

科研

发育生物学

Sequestration of Mouse Myoblasts onto Activated Concanavalin-A Beads

将小鼠成肌细胞隔离到激活的刀豆球蛋白-A 珠上

Genomic Purification and Library Preparation of Tagmented Mouse Myoblast DNA for Epigenetic Profiling

标记小鼠成肌细胞DNA的基因组纯化和文库制备用于表观遗传学分析