sequestration of mouse myoblasts onto activated concanavalin-a beads

CUT &#38; Tag Kullanarak Fare Miyoblastlarında Genomik Histon İşaretlerinin Profillenmesi

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.

Yazar Spotlight: Kas Kök Hücre Çalışmalarında Zorluklar ve Yenilikler Arasında Gezinmek

Fare Miyoblast Araştırmalarında Hedefler Altında Bölünme ve Etiketleme (CUT &#38; Tag) Testinin Kullanılması

Fare Miyoblastlarının Aktive Edilmiş Concanavalin-A Boncukları Üzerine Tutulması

Başlamak için, 1,5 mililitrelik bir santrifüj tüpüne 30 mikrolitre Concanavalin-A boncuğu ekleyin. Boncuklara 300 mikrolitre bağlayıcı tampon pipetleyin. İyice karıştırmak için tüpü birkaç kez ters çevirin, ardından tüpü manyetik bir rafa yerleştirin.Çözelti berraklaştırıldıktan sonra, süpernatanı pipetleyin. Raftan çıkardıktan sonra tüpe 300 mikrolitre bağlayıcı tampon pipetleyin. Tüpün içeriğini karıştırmak için tekrar ters çevirin.Tüpün içeriğini sekiz PCR tüp şeridinin üç tüpüne eşit olarak dağıtın, ardından çözelti berraklaşana kadar tüp şeridini manyetik rafa yerleştirin. Süpernatanı çıkardıktan sonra, tüplere 10 mikrolitre bağlayıcı tampon pipetleyin ve karıştırın. Hazırlanan boncukları daha fazla deney yapana kadar buzun üzerine yerleştirin.Daha sonra, kültürlenmiş fare miyoblastlarını plakalardan deneyin. Hücrelerin santrifüjlenmesinden sonra, süpernatanı vakumla çıkarmak için vakum kullanın. Ardından peleti PBS'de yeniden askıya alın.Süspansiyonun hücre sayımını gerçekleştirdikten sonra hücreleri santrifüjleyin, ardından mililitre başına 700.000 hücrelik bir hücre yoğunluğu oluşturmak için peleti yeterli hacimde yıkama tamponunda yeniden süspanse edin. Bu hücre süspansiyonundan 100 mikrolitreyi PCR tüp şeridinin her bir tüpüne pipetleyin ve iyice karıştırın, ardından Concanavalin-A boncuklarının hücreleri yakalamasına izin vermek için tüp şeridini oda sıcaklığında 10 dakika boyunca bir tahterevalli hareket çalkalayıcıya yerleştirin. Süpernatanı pipetlemeden önce tüplerin içeriğini manyetik bir rafta bir kez daha netleştirin.Son olarak, Concanavalin-A boncuğunun hücrelerle bağlanmasının etkinliğini değerlendirmek için süpernatanı mikroskop altında gözlemleyin.

sequestration-of-mouse-myoblasts-onto-activated-concanavalin-a-beads

Research

JoVE Journal

Developmental Biology

Sequestration of Mouse Myoblasts onto Activated Concanavalin-A Beads

51 Görüntüleme. Başlamak için, 1,5 mililitrelik bir santrifüj tüpüne 30 mikrolitre Concanavalin-A boncuğu ekleyin. Boncuklara 300 mikrolitre bağlayıcı tampon pipetleyin. İyice karıştırmak için tüpü birkaç kez ters çevirin, ardından tüpü manyetik bir rafa yerleştirin.Çözelti berraklaştırıldıktan sonra, süpernatanı pipetleyin. Raftan çıkardıktan sonra tüpe 300 mikrolitre bağlayıcı tampon pipetleyin. Tüpün içeriğini karıştırmak için tekrar ters çevirin.

Video: Fare Miyoblastlarının Aktive Edilmiş Concanavalin-A Boncukları Üzerine Tutulması

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.

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.

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 ...