我们感谢阿纳斯塔西娅·班沃斯（Anastasia Bannwarth）提供出色的技术援助。我们感谢IGBMC动物舍设施、细胞培养、小鼠临床研究所（ICS，Illkirch，法国）、成像、电子显微镜、流式细胞术和GenomEast平台，“法国Génomique”联盟（ANR-10-INBS-0009）的成员。
作为斯特拉斯堡大学、CNRS 和 Inserm 的 ITI 2021-2028 计划的一部分，跨学科专题研究所 IMCBio 的这项工作得到了 IdEx Unistra （ANR-10-IDEX-0002） 和 SFRI-STRAT'US 项目 （ANR 20-SFRI-0012） 和 EUR IMCBio （ANR-17-EURE-0023） 在法国未来投资计划的框架下的支持。INSERM、CNRS、Unistra、IGBMC、Agence Nationale de la Recherche （ANR-16-CE11-0009， AR2GR）、AFM-Téléthon 战略计划 24376（给 D.D.）、INSERM 青年研究员补助金（给 D.D.）、ANR-10-LABX-0030-INRT 和由 ANR 根据 Investissements d'Avenir 框架计划 （ANR-10-IDEX-0002-02） 管理的法国国家基金提供了额外的资金。J.R.得到了法国阿莱曼德大学和高等研究与创新部的CDFA-07-22计划的支持，以及IGBMC研究协会（ARI）的K.G.支持。

小鼠被饲养在经认可的动物舍中，符合国家动物护理指南（欧盟委员会指令86/609/CEE;法国第87-848号法令）关于使用实验动物进行研究。根据 APAFIS 编号 #22281 下的 2010/63/EU 指令，将预期的操作提交给伦理委员会（Com'Eth，斯特拉斯堡，法国）和法国研究部 （MESR） 进行伦理评估和授权。
1.通过荧光激活细胞分选（FACS）制备用于分离卫星细胞的细胞悬液（图1）
<ol><li>肌肉组织的分离<ol><li>使用清洁剂对肌肉解剖工具（包括镊子、手术刀和剪刀）进行去污（表 1），并用蒸馏水彻底冲洗。</li>
		<li>准备两个 2 mL 管（表 1），每个管含有 1 mL 肌肉分离缓冲液，并将它们放在冰上以收集收获的肌肉。</li>
		<li>通过CO2 窒息后宫颈脱位处死两只10周龄的C57 / Bl6J雄性小鼠。在每只小鼠上喷洒70%乙醇。用镊子从后肢剥离皮肤。解剖股骨，胫骨和腓骨周围的所有肢体肌肉（每只小鼠约1mg肌肉）。 注意：卫星细胞数量在 15 周龄后减少。</li>
		<li>将收获的肢体肌肉放入含有1mL肌肉分离缓冲液的2mL管中，该管在步骤1.1.2中制备。按照相同的步骤从第二只鼠标收集肌肉。用剪刀在冰上切碎收获的肌肉，直到获得小于 1 毫米的3 个碎片。 注：肌肉主要按描述12收集和切碎。按照步骤1.2或1.3进行组织消化。</li>
	</ol></li>
	<li>用胶原酶消化组织<ol><li>通过将切碎的肌肉悬浮液倒入含有18mL肌肉分离缓冲液（补充有5mL [5U / mL]分散酶和5mg I型胶原酶）的50mL管（表1）中，转移两只小鼠的碎肌肉悬浮液（表1）。</li>
		<li>紧紧关闭试管并用实验室薄膜密封（表1）。将其水平置于37°C，100rpm的振荡水浴（表1）中30分钟。</li>
		<li>30分钟后，加入5mg的I型胶原酶。将试管在37°C，100rpm的振荡水浴中再搅拌30分钟。</li>
		<li>消化后，用 10 mL 移液管上下移液肌肉悬液 10 次，以提高解离效率。在4°C下以400× g 离心5分钟。在管子底部可以看到一个透明的颗粒。使用 10 mL 移液管弃去上清液，在试管中留下 5 mL 培养基。 注意：离开培养基有助于避免对细胞造成压力。加入 10 mL 新鲜肌肉分离缓冲液，并用 10 mL 移液管上下移液，重悬沉淀。</li>
		<li>将 100 μm、70 μm 和 40 μm 细胞过滤器（每种一种）（表 1）放在开放的 50 mL 管上。将悬浮液移液到连续的细胞过滤器（100μm，70μm，40μm）上，并将流出物收集到50mL管中，其中包含40μm以下的细胞。</li>
		<li>将悬浮液在4°C下以400× g 离心5分钟。使用 10 mL 移液管弃去上清液，直到剩余 2 mL，然后使用 0.2-1 mL 移液管直到剩余 100-200 μL。将沉淀重悬于2mL红细胞裂解缓冲液中（表2）。在冰上孵育3分钟。</li>
		<li>在4°C下以400× g 离心5分钟，并使用20-200μL移液管弃去上清液。将细胞重悬于100μL冷FACS缓冲液中（表2）。放在冰上。</li>
	</ol></li>
	<li>使用 Liberase 低热溶素 （TL） 酶进行组织消化的替代方法<ol><li>按照步骤 1.1 中的说明进行操作。用于组织分离。</li>
		<li>对于Liberase介导的组织解聚，在2mL罗斯威尔公园纪念研究所（RPMI）分离缓冲液（表2）中收获肌肉，而不是步骤1.1.2中描述的肌肉分离缓冲液。</li>
		<li>通过将切碎的肌肉悬浮液从两只小鼠倒入含有18mL RPMI分离缓冲液的50mL管（表1）中，补充有300或600μL的Liberase TL，浓度为5mg / mL（表1）（即，分别为0.083mg / mL和0.167mg / mL终浓度）13。</li>
		<li>紧紧关闭试管并用实验室薄膜密封（表1）。将其水平置于37°C，100rpm的振荡水浴（表1）中30分钟。</li>
		<li>消化后，用 10 mL 移液管将肌肉悬液上下移液 10 次，以解离并提高解离效率。</li>
		<li>在4°C下以400× g 离心5分钟。在管子底部可以看到一个透明的颗粒。使用 10 mL 移液管弃去上清液，在试管中留下 5 mL 培养基。离开培养基有助于避免对细胞造成压力。加入 10 mL 新鲜的 RPMI 分离缓冲液，并用 10 mL 移液管上下移液，重悬沉淀。</li>
		<li>将 100 μm、70 μm 和 40 μm 细胞过滤器（每种一种）（表 1）放在开放的 50 mL 管上。</li>
		<li>将悬浮液移液到连续的细胞过滤器（100μm，70μm，40μm）上，并将流出物收集到50mL管中，其中含有40μm以下的细胞。</li>
		<li>将悬浮液在4°C下以400× g 离心5分钟。使用 10 mL 移液管弃去上清液，直到剩余 2 mL，然后使用 0.2-1 mL 移液管直到剩余 100-200 μL。</li>
		<li>将沉淀重悬于2mL红细胞裂解缓冲液中（表2）。在冰上孵育3分钟。</li>
		<li>在4°C下以400× g 离心5分钟，并使用20-200μL移液管弃去上清液。</li>
		<li>将细胞重悬于100μL冷FACS缓冲液中（表2）。放在冰上。</li>
	</ol></li>
	<li>制备用于FACS分离的细胞悬液<ol><li>将步骤 1.2.12 中获得的 10 μL 细胞悬液转移到新鲜的 1.5 mL 管中。该样品将构成未染色的对照或阴性对照（图2）。加入 190 μL FACS 缓冲液，转移到 5 mL 试管中（表 1），并储存在冰上。</li>
		<li>将步骤1.2.12中获得的剩余90μL细胞悬液在4°C下以400× g 离心5分钟，并使用移液管（20-200μL吸头体积）弃去上清液。将细胞与在室温 （RT） 下在无血清 Dulbecco 改良的 Eagle 培养基 （DMEM） 中稀释的 400 μL 可固定活力染色剂（表 3）孵育 15 分钟。</li>
		<li>通过在4°C下以400× g 离心5分钟洗涤细胞，并加入100μLFACS缓冲液。轻轻地倒置试管三次，并在4°C下以400× g 再次离心5分钟。</li>
		<li>在离心期间，制备 100 μL 与荧光基团偶联并针对 CD11b、CD31、CD45、TER119、CD34、ITGA7 和 CXCR4 的一抗预混液（表 3），在 FACS 缓冲液中稀释。</li>
		<li>在4°C下以400× g 离心5分钟，使用移液管（20-200μL尖端体积）弃去细胞上清液，并加入100μL抗体混合物。轻轻地将管子倒置三次。不要涡旋。在黑暗的冰上孵育30分钟。</li>
		<li>在4°C下以400× g 离心5分钟。 使用 20-200 μL 移液管弃去上清液，并加入 500 μL 1x 磷酸盐缓冲盐水 （PBS） 洗涤细胞。轻轻地将管子倒置三次。在4°C下以400× g 重新离心5分钟，并使用20-200μL移液管弃去上清液。</li>
		<li>将细胞沉淀重悬于500μL流式细胞仪缓冲液中，并将悬浮液转移到5mL试管中。 注意：从Liberase消化获得的细胞悬液以相同的方式处理。</li>
	</ol></li>
	<li>通过FACS选择卫星小区<ol><li>短暂涡旋细胞悬液（2-5秒），并在配备100μm喷嘴的流式细胞仪上处理细胞（表1）。</li>
		<li>根据步骤1.4.1中存储的未染色样品确定各种门尺寸（图2）。</li>
		<li>用 1 mL 纯胎牛血清 （FCS） 包被 5 mL 试管以改善细胞收集，并加入 500 μL FACS 缓冲液。</li>
		<li>将未染色的样品与抗体标记的样品交换。</li>
		<li>根据前向散射区（FSC-A）和侧向散射区（SSC-A）选择感兴趣的群体（图3A），并去除具有FSC-A和前向散射高度（FSC-H）的双峰细胞（图3B）14。</li>
		<li>鉴定具有可固定活力染色阴性染色的活细胞（图3C）。</li>
		<li>选择CD31，CD45，TER119和CD11b的阴性细胞（图3D）。</li>
		<li>为了鉴定卫星细胞，首先选择CD34和ITGA7阳性的细胞（图3E），然后在CD34和ITGA7选择的群体中选择CXCR4阳性细胞（图3F）。</li>
		<li>将选定的细胞（40,000至80,000个细胞，根据制备质量）收集在含有500μLFACS缓冲液的5mL包被管中。</li>
	</ol></li>
</ol>2. 组织培养中分离群体的验证
<ol><li>用水凝胶涂覆幻灯片<ol><li>在 12 mL 无血清 DMEM/F12 培养基中稀释 280 μL 纯水凝胶人胚胎干细胞 （hESC） 合格基质（表 1）。</li>
		<li>用水凝胶溶液涂覆腔室载玻片（表1），并在4°C下孵育过夜。</li>
		<li>第二天，在细胞接种前将腔室载玻片在37°C和5%CO2 下孵育1小时。</li>
	</ol></li>
	<li>细胞生长和分化<ol><li>每孔铺出约20,000个细胞，从步骤1.5.9获得，并在生长培养基（表2）中生长5天。使用明场显微镜拍摄相差图像（图4A），然后对其进行处理以进行免疫荧光分析，以确保制备的质量（图4B）。</li>
		<li>为了诱导肌生成，在肌源性培养基（表2）中再生长步骤2.2.1中扩增的卫星细胞7天。在处理它们进行免疫荧光分析之前，使用明场显微镜（图4C）拍摄相差图像，以确保制备的质量（图4D）。</li>
	</ol></li>
	<li>免疫细胞荧光分析<ol><li>轻轻除去培养基，用100μL的1x PBS洗涤在腔室载玻片上培养的细胞两次，并在室温下用100μL的4%多聚甲醛（PFA）固定1小时。 注意：此步骤应小心执行。腔室中应始终保留少量培养基，以防止对细胞施加压力，并且应将PBS倒入腔室壁。</li>
		<li>用 100 μL 的 1x PBS 洗涤细胞 3 次，并补充有 0.1% 吐温 20 （PBST） 以透化细胞膜。</li>
		<li>通过在室温下在补充有 5% FCS （PBST-FCS） 的 100 μL 1x PBST 中孵育 1 小时来阻断非特异性信号。</li>
		<li>将细胞与100μL抗PAX7和抗肌萎缩蛋白（DMD）抗体（在1x PBST-FCS中稀释）的预混液在4°C下孵育过夜，以分别检测卫星细胞和肌纤维。</li>
		<li>用 100 μL 的 1x PBST 洗涤细胞 3 次，并用 100 μL 山羊抗小鼠 Cy3 或山羊抗兔 Alexa 488 二抗（表 3）在室温下稀释在 1x PBST-FCS 中孵育 1 小时。</li>
		<li>使用供应商提供的设备将腔室孔与载玻片分离，加入 20 μL 含 4′，6-二脒基-2-苯基吲哚 （DAPI） 的水性封固剂，并用盖玻片盖住载玻片（表 1）。</li>
		<li>用共聚焦显微镜观察并捕获染色细胞的图像。</li>
		<li>使用图像分析软件处理图像（图4B，D）。</li>
	</ol></li>
</ol>3. CUT&RUN分析
<ol><li>用于FACS分离卫星细胞的CUT&RUN分析的样品制备 注意：CUT&RUN基本上如所述执行10,15.缓冲液组成见 表2.<ol><li>对于CUT&RUN测定，使用方法1步骤1.5.9中获得的大约40,000个细胞，每个样品/抗体必须进行测试。</li>
		<li>将FACS分离的卫星细胞在室温下以500× g 离心10分钟，然后使用移液管（20-200μL吸头体积）弃去上清液。</li>
		<li>用1mL 1x PBS洗涤细胞，在室温下以500× g 离心5分钟，使用移液管（0.2-1mL尖端体积）弃去上清液，并将它们重悬于1mL冷核提取缓冲液中（表2）。在冰上孵育20分钟。</li>
		<li>在孵育过程中，准备一个含有 850 μL 冷结合缓冲液的 1.5 mL 管（表 2），每个样品加入 20 μL 刀豆球蛋白 A 包被的磁珠（表 1）。</li>
		<li>使用磁架用 1 mL 冷结合缓冲液洗涤珠子两次（表 1）。对于整个过程中的每次洗涤或缓冲液更换，让珠子在磁架上的管侧积聚5分钟，然后用移液管（0.2-1mL吸头体积）除去清除的上清液。然后，轻轻地重悬于 300 μL 冷结合缓冲液中。</li>
		<li>将细胞核在4°C下以600× g 离心5分钟，并将其轻轻重悬于600μL核提取缓冲液中。将600μL提取的细胞核与300μL刀豆球A珠浆轻轻混合，并在4°C下孵育10分钟。</li>
		<li>如步骤3.1.4所述，使用磁性架除去上清液，并用1mL冷封闭缓冲液轻轻重悬珠结合的细胞核（表2）。在室温下孵育 5 分钟。</li>
		<li>使用磁性架除去上清液，并用 1 mL 冷洗缓冲液洗涤珠结合的细胞核两次（表 2）。在第二次洗涤过程中，将珠子结合的细胞核平均分成 1.5 mL 管。在以下步骤中，每根试管都将用特异性抗体处理。 注：在本例中，将 250 μL 微珠结合的细胞核分成四个 1.5 mL 试管。</li>
		<li>如步骤3.1.4所述，使用磁性架分离上清液，并用移液管吸出。用特异性一抗（表3）或在250μL冷洗缓冲液中稀释的其他物种（此处为兔子）的IgG轻轻重悬细胞核/微珠复合物。在4°C孵育过夜，轻轻搅拌。 注意：此处使用的抗体针对 AR、H3K4me2 和 H3K27ac。</li>
		<li>如步骤3.1.4所述，用磁性架除去上清液，用1mL冷洗缓冲液洗涤珠结合的细胞核两次，并重悬于100μL冷洗缓冲液中。</li>
		<li>在每个冷洗缓冲液样品中，以 1.4 ng/μL 的浓度稀释蛋白 A-微球菌核酸酶，每份样品 100 μL。</li>
		<li>将100μL蛋白A-微球菌核酸酶加入到3.1.11中获得的100μL样品中，并在4°C下搅拌孵育1小时。</li>
		<li>如步骤3.1.4所述，用磁性架除去上清液，用1mL冷洗缓冲液洗涤两次，并将珠结合的细胞核重悬于150μL冷洗缓冲液中。</li>
		<li>为了开始DNA切割，向150μL样品中加入3μL的100mM CaCl2 ，通过轻弹快速混合，并在冰上孵育30分钟。通过加入150μL终止缓冲液终止反应，并在37°C下孵育20分钟以消化RNA并释放DNA片段。</li>
		<li>对于DNA提取，将样品在4°C下以16,000× g 离心5分钟。</li>
		<li>将上清液转移到新的微量离心管中，弃去沉淀和珠子。</li>
		<li>加入 3 μL 10% 十二烷基硫酸钠 （SDS） 和 2.5 μL 20 mg/mL 蛋白酶 K。在70°C孵育10分钟（不摇晃）。</li>
		<li>加入300μL苯酚/氯仿/异戊醇，涡旋，转移到2mL锁相管中（在16,000×g下预纺5分钟），并在4°C下以16,000×g离心5分钟。</li>
		<li>向同一管中加入300μL氯仿，并在4°C下以16,000× g 离心5分钟。 用移液管（0.2-1 mL 吸头体积）收集上清液 （~300 μL） 并转移到新的 1.5 mL 管中。</li>
		<li>加入 1 μL 糖原（浓度为 20 mg/mL）。</li>
		<li>加入750μL的100%乙醇，并在-20°C下沉淀过夜。</li>
		<li>通过在4°C下以16,000×g离心15分钟来沉淀DNA。 用1mL 100%乙醇洗涤沉淀，以16,000×g离心5分钟，弃去上清液，以16,000×g离心30秒，并用移液管（20-200μL吸头体积）除去液体。</li>
		<li>将沉淀风干~5分钟。重悬于 25 μL 1 mM Tris-HCl （pH 8） 和 0.1 mM 乙二胺四乙酸 （EDTA;pH 8） 中。</li>
	</ol></li>
	<li>生物信息学分析<ol><li>从免疫裂解的 DNA 制备文库，并在基因组平台的帮助下将它们测序为双端 100 bp reads，如所述16。</li>
		<li>删除与 ENCODE 黑名单区域 （V2） 重叠的读段，并将剩余的读段分成两组：片段大小 <120 bp（无核小体，通常用于转录因子）和片段大小 >150 bp（有核小体，通常用于组蛋白标记）。使用 Bowtie 2 （v2.3.4.3）17 定位到 mm10 参考基因组。</li>
		<li>使用 bamCoverage 生成 bigwig 文件（deeptools 3.3.0：bamCoverage --normalizeUsing RPKM --binSize 20）。</li>
		<li>保留唯一映射的读取以供进一步分析。</li>
		<li>使用 genomeCoverageBed （bedtools v2.26.0） 生成原始床图文件。</li>
		<li>使用 SEACR 1.3 算法（严格选项）进行峰值调用。以UCSC贝图格式加载目标数据基图文件，省略包含零信号的区域，并加载对照（IgG）数据基图文件，以生成峰调用18的经验阈值。</li>
		<li>使用 deeptools 进行 Pearson 相关性分析，以确定样本之间的相似性19.使用命令行 multiBamSummary bins --bamfiles file1.bam file2.bam -o results.npz，后跟 plotCorrelation -in results.npz --corMethod pearson --skipZeros --plotTitle “Pearson Correlation of Read Counts” --whatToPlot heatmap --colorMap RdYlBu --plotNumbers -o heatmap_PearsonCorr_readCounts.png --outFileCorMatrix PearsonCorr_readCounts.tab。</li>
		<li>使用床图文件和从SEACR获得的床文件峰，使用IGV20 可视化全基因组强度分布。</li>
		<li>使用 HOMER 进行峰注释和基序搜索21.</li>
		<li>最后，使用 ChIP-Atlas Peak 浏览器将数据集与先前发布的数据集进行比较，以在 IGV 上可视化它们，和/或使用 SEACR 生成的床文件作为输入数据集进行富集分析22。</li>
	</ol></li>
</ol>

从小鼠肢体肌肉中分离卫星细胞和流式细胞术分析

<ol>
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作者声明他们没有相互竞争的经济利益。

本研究报告了一种标准化、可靠且易于执行的小鼠卫星细胞分离和培养方法，以及通过 CUT&RUN 方法评估转录调控。
该协议涉及几个关键步骤。首先是肌肉破坏和纤维消化，以确保收集到的大量细胞。尽管酶浓度增加，但获得的活细胞比使用方案 1 更多。卫星细胞表达各种膜蛋白的特定模式。为了提高分选的严格性，我们使用了先前描述的阴性（CD31、TER119、CD45 和 CD11b）和阳?...

骨骼横纹肌平均占人体总重量的 40%1。肌肉纤维在受伤时表现出显着的再生能力，这可以通过新形成的肌细胞的融合和取代受损肌纤维的新肌纤维的产生来描述2。1961年，亚历山大·毛罗（Alexander Mauro）报道了一组单核细胞，他称之为卫星细胞3。这些干细胞表达转录因子配对盒 7 （PAX7），位于基底层和肌纤维肌节之间4。据报道，它们表达分化簇 34（CD34;一种造血、内皮祖细胞和间充质干细胞标志物）、整合素 α7（ITGA7;一种平滑、心脏和骨骼肌标志物）以及 C-X-C 趋化因子受体 4 型（CXCR4;一种淋巴细胞、造血和卫星细胞标志物）5。在基础条件下，卫星细胞位于特定的微环境中，使它们保持静止状态6。肌肉损伤后，它们被激活、增殖并发生肌生成7。然而，它们只占肌肉细胞总数的一小部分，它们的全基因组分析特别具有挑战性，尤其是在生理环境下（占总细胞的<1%）。
已经描述了从卫星细胞中分离染色质的各种方法，包括染色质免疫沉淀，然后进行大规模平行测序 （ChIP-seq） 或靶标切割和标记 （CUT&Tag） 实验。然而，这两种技术存在一些重要的局限性，这些局限性仍然没有受到挑战。事实上，ChIP-seq 需要大量的起始材料来产生足够的染色质，其中很大一部分在超声处理步骤中丢失。CUT&Tag 更适合低细胞数，但由于 Tn5 转座酶活性，与 ChIP-seq 相比，CUT&Tag 产生更多的脱靶切割位点。此外，由于该酶对开放染色质区域具有高亲和力，因此 CUT&Tag 方法可能优先用于分析与基因组活跃转录区域相关的组蛋白修饰或转录因子，而不是沉默的异染色质 8,9。
这里介绍的是一个详细的方案，允许通过FACS分离小鼠肢体肌肉卫星细胞，以便在靶标下进行切割，并使用核酸酶（CUT&RUN）10,11分析释放。各个步骤涉及组织的机械破坏、细胞分选和细胞核分离。通过对共价组蛋白修饰和转录因子进行CUT&RUN分析，证明了该方法在制备活细胞悬液方面的效率。分离细胞的质量使得所描述的方法对于制备染色质特别有吸引力，该染色质可以忠实地捕获天然基因组占有状态，并且可能适用于捕获特定位点（4C-seq）或全基因组水平（Hi-C）的高通量测序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microtube</td>
			<td>Eppendorf</td>
			<td>2080422</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microtube</td>
			<td>Star Lab</td>
			<td>S1620-2700</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL tubes</td>
			<td>CORNING-FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-AR</td>
			<td>abcam</td>
			<td>ab108341</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD11b</td>
			<td>eBioscience</td>
			<td>25-0112-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD31</td>
			<td>eBioscience</td>
			<td>12-0311-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>eBioscience</td>
			<td>48-0341-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>eBioscience</td>
			<td>12-0451-83</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CXCR4</td>
			<td>eBioscience</td>
			<td>17-9991-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DMD</td>
			<td>abcam</td>
			<td>ab15277</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3K27ac</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3K4me2</td>
			<td>Active Motif</td>
			<td>39141</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ITGA7</td>
			<td>MBL</td>
			<td>k0046-4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PAX7</td>
			<td>DSHB</td>
			<td>AB_528428</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-TER119</td>
			<td>BD Pharmingen TM</td>
			<td>553673</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads</td>
			<td>Polysciences</td>
			<td>86057-3</td>
			<td>BioMag&#174;Plus Concanavalin A</td>
		</tr>
		<tr>
			<td>Cell Strainer 100 &#181;m</td>
			<td>Corning&#174;&#160;</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#181;m</td>
			<td>Corning&#174;&#160;</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#181;m</td>
			<td>Corning&#174;&#160;</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 1</td>
			<td>Eppendorf</td>
			<td>521-0011</td>
			<td>Centrifuge 5415 R</td>
		</tr>
		<tr>
			<td>Centrifuge 2</td>
			<td>Eppendorf</td>
			<td>5805000010</td>
			<td>Centrifuge 5804 R</td>
		</tr>
		<tr>
			<td>Chamber Slide System&#160;</td>
			<td>ThermoFischer</td>
			<td>171080</td>
			<td>Syst&#232;me Nunc&#8482; Lab-Tek&#8482; Chamber Slide</td>
		</tr>
		<tr>
			<td>Cleaning agent</td>
			<td>Sigma&#160;&#160;</td>
			<td>SLBQ7780V</td>
			<td>RNaseZAPTM</td>
		</tr>
		<tr>
			<td>Collagenase, type I&#160;</td>
			<td>Thermo Fisher</td>
			<td>17100017</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>STEMCELL technologies</td>
			<td>7913</td>
			<td>5 U/mL</td>
		</tr>
		<tr>
			<td>DynaMag&#8482;-2 Aimant</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Stain</td>
			<td>BD Biosciences</td>
			<td>565388</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD FACSAria&#8482; Fusion Flow Cytometer</td>
			<td>23-14816-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount G with DAPI</td>
			<td>Invitrogen</td>
			<td>00-4959-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Genome browser&#160;</td>
			<td>IGV</td>
			<td></td>
			<td>http://software.broadinstitute.org/software/igv/</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>Corning&#174;&#160;</td>
			<td>354277</td>
			<td>Matrigel hESC qualified matrix</td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>Image J&#174;</td>
			<td></td>
			<td>V 1.8.0</td>
		</tr>
		<tr>
			<td>Laboratory film</td>
			<td>Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td>PARAFILM&#174; M</td>
		</tr>
		<tr>
			<td>Liberase LT</td>
			<td>Roche</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>2370</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer&#160;</td>
			<td>Illumina Hiseq 4000</td>
			<td>SY-401-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>Bioblock Scientific polytest 20</td>
			<td>18724</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient protocol for cut&amp;run analysis of facs-isolated mouse satellite cells

小细胞群的全基因组分析是研究的主要制约因素，特别是在干细胞领域。这项工作描述了一种有效的协议，用于从肢体肌肉（一种结构蛋白含量高的组织）中分离卫星细胞的荧光激活细胞分选（FACS）。通过在补充有分散酶和 I 型胶原酶的培养基中切碎来机械地破坏成年小鼠的解剖肢体肌肉。消化后，匀浆通过细胞过滤器过滤，细胞悬浮在FACS缓冲液中。用可固定的活力染色测定活力，并通过FACS分离免疫染色的卫星细胞。用 Triton X-100 裂解细胞，释放的细胞核与刀豆球蛋白 A 磁珠结合。将细胞核/微珠复合物与针对目标转录因子或组蛋白修饰的抗体一起孵育。洗涤后，将细胞核/微珠复合物与蛋白 A-微球菌核酸酶一起孵育，并用 CaCl2 开始染色质裂解。提取DNA后，建立文库并进行测序，通过生物信息学分析获得全基因组转录因子结合和共价组蛋白修饰图谱。获得的各种组蛋白标记的峰表明，结合事件对卫星细胞具有特异性。此外，已知基序分析显示转录因子 通过其 同源反应元件与染色质结合。因此，该方案适用于研究成年小鼠肢体肌肉卫星细胞中的基因调控。

通过结合Gunther等人（以下简称方案1）12 和Liu等人23 （以下简称方案2）的方案从小鼠骨骼肌中分离卫星细胞。由于当使用方案1中提出的胶原酶和分散酶的浓度时，在消化后观察到未消化的肌纤维，因此增加酶的量以改善肌纤维解离，如步骤1.2.1和1.2.3所述。如方案2所示，将样品在水浴中轻轻搅拌以保持细胞活力。如方案1所述，我们通过细胞过滤器进行过滤，并...

Watch this Scientific Journal Video about Cistrome Analysis in Mouse Muscle Stem Cells at JoVE.com

作者聚焦：小鼠肌肉干细胞中的 Cistrome 分析 

这里介绍的是一种有效的方案，用于小鼠肢体肌肉卫星细胞的荧光激活细胞分选（FACS）分离，适用于研究肌肉纤维中的转录调控，方法是在靶标下切割并使用核酸酶（CUT&RUN）释放。

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

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

20多年来，我们的实验室一直对核受体的病理生理作用感兴趣。为了破译它们的体内功能，我们设计了小鼠模型，允许对它们的表达进行空间和时间控制。同时，我们开发了尖端技术来研究它们在小鼠组织中的分子功能。我们的科学兴趣最近促使我们确定各种组织（包括骨骼肌）中的类固醇和类固醇受体信号传导。我们最近证明，分化骨骼肌细胞的肌纤维中的雄激素受体有助于其收缩和代谢功能。我们最近的研究结果提供了对骨骼肌病理生理动力学的更深入理解，这对于开发肌肉相关疾病的有效治疗方法至关重要。我们开发了该协议，以便能够在体内对肌肉干细胞进行雌性肿瘤分析。所有以前的协议都使用体外模式，这完全将研究与整个生物体背景分离。除了低成本和省时之外，该协议还为研究骨骼肌祖细胞中的转录因子募集和染色质景观提供了有效的实验环境。通过该协议制备的染色质提供了卫星细胞中AR雌激素的首次全基因组分析，并将促进未来基因调控的研究。在众多研究中，我们未来的目标是将这些研究扩展到其他氧质类固醇受体，并探索它们在骨骼肌再生过程中参与的各种细胞类型中的特定共调节因子。

an-efficient-protocol-for-cut-run-analysis-facs-isolated-mouse

Research

JoVE Journal

Developmental Biology

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

2.1K 次观看.  Université de Strasbourg. 20多年来，我们的实验室一直对核受体的病理生理作用感兴趣。为了破译它们的体内功能，我们设计了小鼠模型，允许对它们的表达进行空间和时间控制。同时，我们开发了尖端技术来研究它们在小鼠组织中的分子功能。我们的科学兴趣最近促使我们确定各种组织（包括骨骼肌）中的类固醇和类固醇受体信号传导。我们最近证明，分化骨骼肌细胞的肌纤维中的雄激素受?...

视频: 作者聚焦：小鼠肌肉干细胞中的 Cistrome 分析 

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

科研

发育生物学

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...