Name: 使用FACS分选的小鼠卫星细胞的核酸酶分析在靶标下进行切割和释放
Uploaded: 2023-07-07T14:20:00
Description: Watch this Scientific Journal Video about Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells at JoVE.com

cleavage under targets and release using nuclease analysis of facs-sorted mouse satellite cells

从小鼠肢体肌肉中分离卫星细胞和流式细胞术分析

Skeletal striated muscles represent on average 40% of the weight of the total human body1. Muscle fibers exhibit a remarkable capacity for regeneration upon injury, which is described by the fusion of newly formed myocytes and the generation of new myofibers that replace the damaged ones2. In 1961, Alexander Mauro reported a population of mononuclear cells that he termed as satellite cells3. These stem cells express the transcription factor paired box 7 (PAX7), and are located between the basal lamina and the sarcolemma of muscle fibers4. They were reported to express the cluster of differentiation 34 (CD34; a hematopoietic, endothelial progenitor and mesenchymal stem cell marker), integrin alpha 7 (ITGA7; a smooth, cardiac and skeletal muscle marker), as well as the C-X-C chemokine receptor type 4 (CXCR4; a lymphocyte, hematopoietic, and satellite cell marker)5. In basal conditions, satellite cells reside in a particular microenvironment that keeps them in a quiescent state6. Upon muscle damage, they become activated, proliferate, and undergo myogenesis7. However, contributing only to a minor fraction of the total number of muscle cells, their genome-wide analyses are particularly challenging, especially under physiological settings (&#60;1% of total cells).
Various methods for chromatin isolation from satellite cells have been described, which involve chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) or cleavage under targets and tagmentation (CUT&#38;Tag) experiments. Nevertheless, these two techniques present some significant limitations that remain unchallenged. Indeed, ChIP-seq requires a high amount of starting material to generate enough chromatin, a large proportion of which is lost during the sonication step. CUT&#38;Tag is more appropriate for low cell number, but generates more off-target cleavage sites than ChIP-seq due to the Tn5 transposase activity. In addition, since this enzyme has a high affinity for open-chromatin regions, the CUT&#38;Tag approach might be preferentially used for analyzing histone modifications or transcription factors associated with actively transcribed regions of the genome, instead of silenced heterochromatin8,9.
Presented here is a detailed protocol that allows the isolation of mouse limb muscle satellite cells by FACS for cleavage under targets and release using nuclease (CUT&#38;RUN)10,11 analysis. The various steps involve the mechanical disruption of tissue, cell sorting, and nuclei isolation. The method&#39;s efficiency, regarding the preparation of a viable cell suspension, was demonstrated by performing CUT&#38;RUN analysis for covalent histone modifications and transcription factors. The quality of isolated cells makes the described method particularly attractive for preparing chromatin that captures the native genomic occupancy state faithfully, and is likely to be suitable for capturing the chromosome conformation in combination with high-throughput sequencing at specific loci (4C-seq) or at genome-wide levels (Hi-C).

作者聚焦：小鼠肌肉干细胞中的 Cistrome 分析 

一种用于FACS分离的小鼠卫星细胞的CUT&amp;RUN分析的有效方案

Since more than 20 years, our lab is interested in the pathophysiological role of nuclear receptors. To decipher their in vivo function, we engineered mouse model allowing the spatial and temporal control of their expression. In parallel, we developed cutting-edge techniques to investigate their molecular function in the mouse tissues.Our scientific interest recently led us to determine secosteroid and steroid receptor signaling in various tissues, including skeletal muscle. We have recently demonstrated that the androgen receptor in myofibers that differentiate cells of the skeletal muscle is instrumental in their contractile and metabolic functions. Our recent findings deliver a deeper understanding of skeletal muscle pathophysiological dynamics that is crucial to develop effective treatments for muscle-associated disorders.We developed this protocol to be able to perform cistrome analysis on muscle stem cells in vivo. All former protocols used in vitro modals, which completely dissociates the investigation from a whole organism context. In addition to being low-cost and time-efficient, this protocol provides an effective experimental setting to study transcription factor recruitment and chromatin landscape in skeletal muscle progenitor cells.Chromatin prepared by this protocol has provided the first genome-wide analysis of AR cistrome in satellite cells, and will facilitate future studies on gene regulation. Among many, our future aim would be to extend these investigations onto other oxosteroid receptors and explore their specific co-regulators in the various cell types involved in the regeneration process in skeletal muscle.

Watch this Scientific Journal Video about Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells at JoVE.com

使用FACS分选的小鼠卫星细胞的核酸酶分析在靶标下进行切割和释放

离心FACS分离的卫星细胞并弃去上清液。用一毫升PBS洗涤细胞，离心，并在冰上的一毫升冷核提取缓冲液中孵育20分钟。将 20 微升刀豆球蛋白 A 包被的磁珠加入含有 850 微升冷结合缓冲液的 1.5 毫升管中。然后使用磁性架，用一毫升冷结合缓冲液洗涤珠子两次，让珠子在管架上的管侧积聚五分钟，然后轻轻地将它们重悬于 300 微升冷结合缓冲液中。接下来，离心细胞核并将它们轻轻重悬于 600 微升核提取缓冲液中。然后将 600 微升提取的细胞核与 300 微升刀豆球蛋白 A 珠浆混合，并在 4 摄氏度下孵育 10 分钟。使用磁架除去上清液后，用一毫升冷封闭缓冲液重悬珠结合的细胞核。再次，除去上清液，用一毫升冷洗缓冲液洗涤珠结合的细胞核两次。分离上清液，用特异性一抗轻轻重悬核珠复合物，并在 4 摄氏度下温育过夜，轻轻搅拌。第二天，除去上清液并洗涤珠子结合的细胞核，然后重悬于100μL冷洗缓冲液中。将 100 微升稀释的蛋白质 A-微球菌核酸酶加入 100 微升珠子结合核中，并在 4 摄氏度下搅拌孵育 1 小时。孵育后，除去上清液并洗涤珠子结合的细胞核两次，然后将它们重悬于150μL冷洗缓冲液中。接下来，加入 3 微升 100 毫摩尔氯化钙，轻弹快速混合，并在冰上孵育 30 分钟。加入 150 微升终止缓冲液终止反应，并在 37 摄氏度下孵育 20 分钟。离心样品并将上清液转移到新的微量离心管中，丢弃沉淀和珠子。然后加入3微升10%十二烷基硫酸钠和2.5微升每毫升20毫克蛋白酶K.倒置混合，在70摄氏度下孵育10分钟。然后加入 300 微升苯酚：氯仿：异戊醇并涡旋，然后转移到两个毫升锁相管中。离心并向同一管中加入300微升氯仿。再次离心并将上清液收集到1.5毫升管中。接下来，加入 1 微升糖原，然后加入 750 微升 100% 乙醇，让 DNA 在零下 20 摄氏度下沉淀过夜。第二天，通过离心沉淀DNA，然后用一毫升100%乙醇洗涤沉淀并离心两次，然后用移液管除去残留的乙醇。将沉淀风干五分钟，并将其重悬于25微升tris-EDTA缓冲液中。H3KME2 和 H3K27AC 围绕 PAC7、ITGA7、LAM2、CXCR4 和 VCAM1 的 TSS 富集。然而，H3K4ME2和H3K27AC在ITGAM、PTPRC、PECAM、CKM或MHY3中没有得到富集。IgG样品几乎没有获得任何信号。图中显示了 HOMER 已知基序分析、AR 峰和卫星细胞以及目标区域的百分比。Seeker 分析揭示了近 500 个峰值得分高于 50 的峰值，其中 200 多个峰值得分高于 100。

Research

JoVE Journal

Developmental Biology

Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...