这项工作得到了韦恩州立大学 Karmanos 癌症研究所的支持, 由国家心脏、肺和血液研究所 (1K22HL126842-01A1) 授予 B.L.K。这项工作利用韦恩州立大学高性能计算网格计算资源 (< https://www.grid.wayne.edu/>)。我们感谢集集 Kurup 对 CARIP 数据分析的帮助。
作者的贡献:
B.L.K. 构思了 CARIP 法, 设计并进行了芯片序列和 CARIP 的实验, 分析了测序数据, 起草了手稿。所有作者都已阅读并批准了本手稿的最终版本。

1. 小鼠 ES 细胞在无饲养条件下的培养。
注意: 小鼠 ES 细胞是传统的在培养基上培养的细胞培养皿上涂有明胶和单层小鼠胚胎成纤维细胞 (MEF), 已 mitotically 灭活 (iMEFs)。然而, MEFs 应在下游表观或表达分析之前被删除, 以防止与 MEF 相关的染色质和 RNA 的污染。
<ol><li>给料层的制备: 在水中加入2毫升0.1% 的明胶, 并在37摄氏度下孵育20-30 分钟, 明胶涂层 a 6 井细胞培养板。</li>
	<li>准备 10% DMEM 高葡萄糖培养基, 含2毫米 l-谷氨酰胺, 10% 胎牛血清 (血清) 和1x 青霉素/链霉素。</li>
	<li>将 MEFs 从 E13.5 隔离到 E14.5 鼠标18、19或从商业供应商处获取。用标准协议19治疗丝裂霉素 C 或伽玛照射 Mitotically 灭活 MEFs。或者, mitotically 灭活 MEFs (iMEFs) 可以从商业供应商那里获得。</li>
	<li>培养 MEFs。预热的 mef 介质在37摄氏度, 解冻冷冻小瓶的 iMEFs 和文化在10% 的血清 MEF 介质37°c 与 5% CO2。板 MEFs 在20万细胞每井6井培养皿。</li>
	<li>在一层饲养细胞上培养 ES 细胞 (iMEFs)。<ol>
<li>12-24 小时后电镀 iMEFs, 预热50毫升的 ES 细胞培养基 (DMEM 高葡萄糖, LIF (10 ng/毫升), 15% ES 细胞合格的血清, 1×细胞培养合格的 2-巯基乙醇, 1×谷氨酰胺, 不必要的氨基酸 (NEAA), 1×青霉素/链霉素), 在37°c使用 5% CO2。</li>
		<li>从含有 iMEFs 的细胞培养皿中吸取培养基, 在2毫升培养基中重新悬浮 ES 细胞 (在37摄氏度解冻后), 并将细胞层板在 iMEFs 上。将培养基放在孵化器中时, 重要的是把盘子移到 "t" 形上, 均匀地分配细胞, 防止聚集到盘子的中心。</li>
	</ol>
</li>
	<li>将 ES 细胞送到无喂食的明胶涂层菜肴中。在它们汇合之前通过 ES 细胞。ES 细胞菌落保持自我更新最好, 如果殖民地是均匀间隔, 而不是汇合。许多 ES 细胞菌落的存在表明细胞密度过高。<ol>
<li>用上面描述的明胶涂上6井培养皿。孵育0.25% 胰蛋白酶-EDTA 在37摄氏度为 ~ 10-15 分钟前温热胰蛋白酶。</li>
		<li>其次, 通过先用1x 磷酸缓冲盐水 (PBS) 冲洗 ES 细胞, 加入1毫升0.25% 胰蛋白酶-EDTA 溶液。等待1-2 分钟的殖民地分离。将菌落与单细胞悬浮液分离, 吹打1毫升, 5 次。</li>
		<li>用显微镜确认 ES 细胞的单细胞分离。要消除反应, 或 "中和" 胰蛋白酶, 添加10% 个血清 MEF 介质。将细胞在室温下的 300 x g 离心成3-5 分钟, 然后将培养基吸干。</li>
		<li>在2毫升培养基 (ES 细胞) 中重新悬浮细胞颗粒, 辅以糖原合酶 kinase-3 (GSK3) 抑制剂 (CHIR99021;GSK3i), 或 2i (MEKi/GSK3i) 条件 (2-3 µM GSK3i: CHIR99021, 1 µM 张光明: PD0325901)。</li>
		<li>从细胞培养皿中吸取明胶, 并将含有 ESCs 的2毫升介质添加到盘中, 并将菜放在37摄氏度孵化器中 (图 1A-d)。双抑制 GSK3i 和张光明促进地面状态幼稚干细胞的 ES 细胞缺乏饲养细胞 (iMEFs) 和没有血清20。ES 细胞应传代三次, 以去除 MEFs, 然后再进行交联。</li>
	</ol>
</li>
</ol>2. 用于芯片和 CARIP 的 ES 细胞交联
<ol><li>用 PBS 洗涤胚胎干细胞, 然后加入0.25% 胰蛋白酶预热37摄氏度。室温下孵化细胞数分钟。避免超过 trypsinization 的 ES 细胞, 这将导致细胞死亡。用显微镜评价单细胞分离。</li>
	<li>如上文所述, 传代 ES 细胞的猝灭胰蛋白酶消化, 在室温下将 300 x g 的细胞离心为3-5 分钟。</li>
	<li>在 2 x 106单元格/mL 中, 重新挂起预热 (37 °c) MEF 介质中的单元格 (10% 个 DMEM)。</li>
	<li>为了交联细胞, 添加37% 甲醛溶液到1% 最终浓度, 并孵化的细胞在一个锥形管 (15 毫升或50毫升) 在37摄氏度, 8 分钟. 在8分钟内多次反转管以达到均匀固定。 注意: CARIP 的固定时间可以进行经验优化。如果固定时间改变, 超声波周期的数量也应该调整。此外, 37 °c 温度提高交联效率相比, 室温交联。固定温度可以进行经验优化。</li>
	<li>要停止固定反应, 加入 1.25 m 甘氨酸 (预冷在4°c) 到浓度 0.125 m. 用旋转器在室温下用转子在 8 rpm 上反转管。在室温下将样品离心 300 x 克, 室温5分钟, 并吸入介质。</li>
	<li>用预冷 PBS (4 °c) 清洗细胞颗粒, 在室温下离心样品 300 x 克, 在室温下吸入 pbs。再用 PBS 冲洗细胞。接下来, 整除 15 x 106单元格, 每15毫升管。</li>
	<li>将细胞颗粒放置在液氮中1分钟, 在干冰上5分钟, 或立即放置在-80 摄氏度。 注: 固定 ES 细胞颗粒可储存6个月, 而不降低下游实验的质量。</li>
</ol>3. 芯片和 CARIP 的染色质超声波
<ol><li>在冰上解冻固定 ES 细胞颗粒。在超声波缓冲器 (2.5 毫升) 中重新悬浮细胞颗粒。对于转录因子或表观遗传调控因子 (蛋白质因素), 在 1x TE、1mM PMSF、1x 蛋白酶抑制剂鸡尾酒中重新悬浮颗粒。对于组蛋白修饰, 重新悬浮在 1x TE, 0.1% SDS, 1mM PMSF, 1x 蛋白酶抑制剂颗粒。 注: 虽然添加 SDS 提高超声波效率, 但它可能不利于评估某些染色质成分或转录因子。</li>
	<li>为染色质免疫沉淀 (芯片), 油脂实验染色质以14-18 周期 (三十年代, 三十年代休息) 在40% 振幅。对于染色质相关的 RNA 免疫沉淀 (CARIP), 减少超声波周期的数量 (8-10 周期)。由于细胞, 固定, 和 sonifier 的变异性, 超声波的条件应该是经验主义的确定。</li>
	<li>补充超声波缓冲器, 28 µL 10% SDS 的蛋白质因子, 28 µL 钠脱氧, 0.28 毫升 10% Triton-X100, 并混合良好。 注意: 将这些组件添加到超声波缓冲区会生成马国贤缓冲区。</li>
	<li>离心样品在 1万 x g 10 分钟在4°c 之前清除染色质。将上清液移至新管, 并进行染色质免疫沉淀。</li>
	<li>另外, 微气泡染色质可以储存在-80 摄氏度, 直到使用。然而, 微气泡染色质在-80 摄氏度的贮存可能导致蛋白质因子芯片质量下降。</li>
</ol>4. 芯片和 CARIP 工艺
<ol><li>将40µL 的蛋白质 A 或 G 磁性珠子添加到1.5 毫升, 低结合管。添加600µL 的 PBS 来清洗珠子。将管置于装有磁铁的机架上, 在室温下孵化管1分钟, 去除 pbs, 并在 pbs 的100µL 中重新悬浮磁珠。</li>
	<li>用磁珠将2-4 µg 的抗体添加到管中。用旋转器在室温下孵化和旋转管40分钟, 以促进对磁性珠子的抗体结合。将管子插入磁力架上, 在室温下孵化1分钟, 然后取出 PBS。</li>
	<li>添加200µL 的 PBS 来清洗磁性珠子。使用 PBS 进行洗涤以去除自由 IgGs。在洗涤步骤之间, 在室温下孵化和旋转管5分钟。把管子贴在磁铁上, 然后取下 PBS。</li>
	<li>Immunoprecipitate 染色质结合蛋白通过孵化微气泡染色质提取物 (4 x 10 6 细胞每芯片), 与磁性珠和旋转样品在4°c (隔夜).</li>
	<li>洗磁珠。用以下缓冲器在室温下孵化样品, 并在每个缓冲器上旋转样品10分钟: 两次与1毫升的马国贤缓冲 (TE (10 毫米 Tric 盐酸, 1 毫米 EDTA) pH 8.0, 1% 海卫 X-100, 0.1% 钠 deocycholate, 0.1% SDS), 两次与1毫升马国贤缓冲 containing 0.3 米氯化钠, 两次与1毫升 LiCl 缓冲器 (0.5% 钠 deocycholate, 0.5% NP40, 0.25 M LiCl), 一次与1毫升的 TE 缓冲器与0.2% 海卫一 X-100, 一次与1毫升的 te。</li>
	<li>芯片的脱交联。在100µL 中重新悬浮磁珠。添加3µL 10% SDS 和5µL 蛋白酶 K (20 毫克/毫升)。在65摄氏度 (隔夜) 孵化样品。 注意: 在这个步骤中, 用 parafilm 或塑料包裹来防止蒸发是很重要的。<ol>
<li>第二天, 把管子倒置几次搅拌。接下来, 用磁铁架将上清液转移到新鲜的管子上。要清洗磁珠, 添加100µL 的 TE 含0.5 米氯化钠, 然后结合两个上清液。</li>
	</ol>
</li>
	<li>CARIP 脱交联。在100µL 中重新悬浮磁珠。添加3µL 10% SDS 和5µL 蛋白酶 K (20 毫克/毫升)。将样品孵化为65摄氏度, 4 至12小时, 在65摄氏度孵化时间需要经过经验确定, 因为细胞类型, 超声波和固定条件等的变异性。<ol>
<li>将管子倒置3-5 次混合, 将管子应用到磁机架上, 然后将上清移到新管上。</li>
		<li>要清洗磁珠, 添加100µL 的 TE 含0.5 米氯化钠, 然后结合两个上清液。 注意: 使用无核酸酶试剂 (e.g, TE 缓冲器, SDS, 等) 是很重要的, 以防止 DNA 或 RNA 在脱交联步骤中丢失. </li>
	</ol>
</li>
	<li>提取 DNA/RNA 使用200µL 的 phenol:chloroform:isoamyl 酒精 (PCI) (25:24:1, v/v)。 注意: 使用制造商的说明, 也可以使用商业工具包提取 RNA。摇动三十年代, 在室温下旋转 1万 x g 10 分钟, 并将上清移至新鲜管。<ol>
<li>为沉淀 DNA/RNA, 添加2µL GlycoBlue, 20 µL 3 米醋酸钠, 600 µL 乙醇。混合反转 ~ 5-8 倍, 并放置在干冰或-80 摄氏度至少1-2 小时的样品。</li>
		<li>用冷冻 (4 °c) 台式离心机将样品在 1万 x g 处离心30分钟。空气干燥颗粒为 < 1 分钟, 并重新暂停它在40µL 的 EB 缓冲器 (10 毫米三 HCl)。对于芯片序列库的准备工作, 请继续步骤6。</li>
	</ol>
</li>
	<li>使用 DNase 从染色质相关的 RNA IP 样品中去除 DNA。将 10x DNase 缓冲区添加到 CARIP 示例中的1x 浓度。添加1µL DNase 10 µg 的 RNA (50 µL 反应)。将样品孵化37摄氏度, 30 分钟. 用 PCI 提取 RNA 样品。并用重悬在无核酸酶H 2 O 中。</li>
</ol>5. 双链 CARIP cDNA 合成
<ol><li>一股 cDNA 的合成。对于反向转录反应, 在为 PCR 机设计的管中设置以下反应: 10.5 µL RNA 和1µL 随机水溶液引物 (3 µg/µL)。</li>
	<li>孵化管在65°c 5 分钟, 以变性的二级结构, 并储存在冰上2分钟. 混合以下组件: 4 µL 的一线缓冲, 2 µL 的 0.1 M 的德勤, 1 µL dNTP, 0.5 RNase。接下来, 对 PCR 管, 添加7.5 µL 的主混合。在25摄氏度孵化管2分钟。添加1µL 的上标 II (200 U/µL) 和孵化如下:25 °c 10 分钟, 42 °c 为50分钟, 70 °c 15 分钟, 并保持在4°c。</li>
	<li>二股 cDNA 合成。把管子放在冰上。接下来, 按顺序添加以下组件:91 µL 二乙基焦碳酸 (DEPC) 处理的水, 30 µL 5x 第二股反应缓冲器, 3 µL dNTP 混合 (10 毫米), 1 µL大肠杆菌dna 连接酶 (10 u/µL), 4 µL 大肠杆菌 dna 聚合酶 (10 u/µL) 和1µL 的大肠杆菌RNase H (2 u/µL)。通过轻轻涡流和孵育16°c 2 小时混合。 注: 通过弹或反转管混合可能不足以彻底混合试剂。</li>
	<li>添加2µL 的 T4 DNA 聚合酶和孵育16°c 5 分钟纯化 cDNA (双绞股, dscDNA) 使用 PCR 纯化试剂盒。洗脱 dscDNA 40 µL 的 EB 缓冲器。</li>
	<li>片段双链 cDNA。剪切 cDNA (dscDNA) 使用水浴 sonifier 和1.5 毫升管的大小为 250-500 bp. 超声波 dscDNA 对于减少碎片大小以便于序列化整个 RNA 转录至关重要。</li>
	<li>当使用一个标准模型的水浴 sonifier, 油脂实验的样品 3×10分钟, 或 4×10分钟。 注: 三周期应用于总 rna 的1µg, 而四周期建议为1-5 µg 的总 rna。在每个超声波循环后, 简单地离心管, 并通过添加新鲜冰保持冷水浴。</li>
	<li>当使用皮 sonifier, 建议使用六周期1-5 µg 的总 RNA 和以下循环条件 (三十年代, 九十年代关闭)。离心管子在2-3 超声波周期以后简要地。超声波的效率可以通过在琼脂糖凝胶 (2%) 上加载一小部分剪切 dscDNA (3-4 µL) 来确定。由于 sonifiers 之间的变异性, 对超声波条件进行经验测试是很重要的。</li>
	<li>按照步骤6中描述的下一代测序执行库准备工作。</li>
</ol>6. 芯片序列与 CARIP 图书馆建设
<ol><li>使用 dna 末端修复试剂盒修复 dna 末端。此套件用于生成钝端 DNA。为此反应, 设置以下在1.5 毫升, 低捆绑的管:34 µL 脱氧核糖核酸, 5 µL 2.5 毫米 dNPTs, 5 µL 10 毫米 ATP, 5 µL 10x 末端修理缓冲器 (5 毫米, 100 毫米醋酸镁, 660 毫米醋酸钾, 300 毫米三醋酸盐, pH 值 7.8) 和1µL 的末端修复酶混合 (T4 核苷酸激酶, T4 DNA 聚合酶)。</li>
	<li>在室温下孵化样品45分钟。</li>
	<li>使用反应清除试剂盒净化 DNA。洗脱最终修复 DNA 32 µL 预热 (65 °c) EB 缓冲器。虽然我们通常不定量 dna 跟随芯片的浓度, 或 rna 跟随 CARIP, 在图书馆准备之前, 建议开始与至少 5-10 ng 芯片脱氧核糖核酸或 CARIP RNA。</li>
	<li>增加 3 ' a ' 的悬臂, 以最终修复 DNA。将组件添加到1.5 毫升, 低结合管:30 µL DNA 从上面, 5 µL 10x 纳布缓冲器 2, 1 µL dATP 从10毫米股票, 11 µL 的 H2O, 3 µL 5 U/µL 克莱诺片段 (3 '-5 ' 外), 并轻轻地混合吹打。</li>
	<li>在37摄氏度孵化样品30分钟。</li>
	<li>使用反应清除试剂盒净化 DNA。洗脱 DNA 在25µL 前预热 (65 °c) EB 缓冲器。</li>
	<li>结扎适配器。在冰上添加以下组件:23 µL dna, 3 µL 10x T4 dna 连接酶缓冲器, 1 µL 退火 PE 或复用适配器 (4 µM), 3 µL T4 DNA 连接酶 (400 U/µL), 并用吹打轻轻混合。 注意: 适配器序列也可以从商业来源获得。 寡核苷酸序列: PE 适配器 5 ' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5 ' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 多路适配器 5 ' P-GATCGGAAGAGCACACGTCT 5 ' ACACTCTTTCCCTACACGACGCTCTTCCGATCT</li>
	<li>在室温下孵化样品30分钟。</li>
	<li>使用预铸 (2%) 琼脂糖凝胶对 DNA 进行尺寸选择。润凝胶10分钟。</li>
	<li>与 250-450 bp 对应的区域消费。 注: 通过切割以上 200 bp 以上的凝胶, unligated 适配器脂肪酸的污染可能性降低。在 PCR 步骤之前删除任何 unligated 适配器是很重要的。</li>
	<li>使用凝胶萃取试剂盒净化 DNA。洗脱在20µL 的 EB 缓冲器中。</li>
	<li>含配对端 (PE) 适配器的结扎 DNA 样品的 PCR 和库纯化。加入 PCR 管中的成分:12 µL 50% 的 DNA 从上面, 12 µL 的水, 25 µL 的主混合 (Phusion HF), 1 µL 的 pe pcr 底漆 1.0, 1 µL 的 pe pcr 底漆2.0。 注意: 寡核苷酸序列也可以从商业来源获得。 寡核苷酸序列: PE PCR 底漆1。0 5 ' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT PE PCR 底漆2。0 5 ' CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT</li>
	<li>含指数 PE 适配器的结扎样品的 PCR 和库净化。添加组件:12 µL 50% 的 DNA 从上面, 12 µL 水, 1 µL pcr 底漆研究所 1.0 (稀释 1:2), 1 µL pcr 底漆研究所 2.0 (稀释 1:2), 和1µL pcr 底漆指数 (稀释 1:2)。 注意: 寡核苷酸序列也可以从商业来源获得。 寡核苷酸序列: 复用 PCR 底漆1。0 5 ' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 复用 PCR 底漆2。0 5 ' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT PCR 引物指数序列 1-12 PCR 底漆, 索引1 5 ' CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTC PCR 底漆, 索引2 5 ' CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTC PCR 底漆, 索引3 5 ' CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTC PCR 底漆, 索引4 5 ' CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTC PCR 底漆, 索引5 5 ' CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTC PCR 底漆, 索引6 5 ' CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTC PCR 底漆, 索引7 5 ' CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTC PCR 底漆, 索引8 5 ' CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTC PCR 底漆, 索引9 5 ' CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTC PCR 底漆, 索引10 5 ' CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTC PCR 底漆, 索引11 5 ' CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTC PCR 底漆, 索引12 5 ' CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTC</li>
	<li>使用以下条件执行 PCR 循环 (变性三十年代在98°c, 18 个周期十年代在98°c, 三十年代在 65°C, 三十年代在72°c, 5 分钟在72°c, 举行在4°c)。 注: PCR 周期的数量应根据经验确定。</li>
	<li>PCR 产品的尺寸选择。在预铸电子凝胶前 (2%) 或自制的2% 凝胶 (琼脂糖) 上运行 DNA, 并在 300-500 bp 区域进行消费。</li>
	<li>使用凝胶萃取试剂盒从凝胶中纯化 DNA。洗脱最终 PCR 产品在20µL 的 EB 缓冲器。</li>
	<li>使用荧光计测量库的浓度。执行下一代测序。</li>
</ol>

<ol>
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作者声明没有利益冲突。

在 ES 细胞中, 芯片序列是评价全球蛋白质-DNA 相互作用 (e. g)、转录因子/组蛋白修饰酶/组蛋白修饰和 DNA 的有效方法, 而新开发的 CARIP 序列协议在审问基因组范围内的 rna 与染色质成分的关联。芯片序列是一种基本的工具, 用于评估 ES 细胞和其他细胞类型的表观景观。芯片序列和 CARIP 库的质量在很大程度上依赖于芯片级抗体。对芯片序列的抗体的适用性可以通过执行芯片 PCR 的经验来确定, ?...

胚胎干细胞的命运决定由细胞外信号和一系列转录调控器之间的通讯调节, 其中包括组蛋白修饰语和组蛋白尾的翻译后修饰。这些相互作用促进染色质可达性和染色质的包装成两种状态之一: 染色质, 是开放和转录活跃的, 染色质, 这是紧凑的, 一般转录不活跃。转录因子与 DNA 序列特异的结合亲和性和表观遗传修饰, euchromatic 区域参与控制基因表达。下一代测序方法, 包括芯片序列1, 在映射全基因组转录网络 (ES 细胞自我更新和干细胞2、3、4) 方面发挥了重要作用. ,5,6。此外, 虽然 rna immunopreciation 其次是下一代测序 (RIP 序列)7对 RNA-蛋白质相互作用的评价表明, DNA 结合蛋白与 rna 相互作用来调节转录事件7,8,9,10,11,12, 很少有研究调查了与染色质12相关的 rna 的全基因组定位, 或者 rna 和组蛋白修饰之间的全球相互作用。长非编码 rna (lncRNAs) 是一种 rna, 它已被发现来调节染色质相关蛋白的活性13,14,15。例如, Xist 是一种 lncRNA, 通过招募阻遏16、17, 在雌性哺乳动物细胞中调节一个 X 染色体的失活。然而, 与染色质相关的 rna 的全谱在很大程度上是未知的。在这里, 我们描述了一个新的协议, 染色质相关的 RNA 免疫沉淀 (CARIP) 其次是下一代测序 (CARIP 序列), 以确定染色质相关 rna 在全基因组的基础上的 ES 细胞, 包括图书馆准备下一代测序, 和芯片序列, 以映射全球占用的组蛋白修饰, 转录因子, 和表观遗传修饰。与其他 RIP 序列方法7不同, CARIP 序列包括交联和超声波步骤, 它允许直接识别与染色质相关的 rna。在一起, 芯片序列是一个强有力的工具, 以确定全基因组范围内的蛋白质-DNA 相互作用, 而 CARIP 是一个强大的方法来调查与染色质成分相关的 rna。

<table>
	<tbody>
		<tr>
			<td>Mouse leukemia inhibitory factor (ESGRO LIF)</td>
			<td>EMD Millipore</td>
			<td></td>
			<td>106 or 107 units</td>
		</tr>
		<tr>
			<td>GSK3&#946; inhibitor CHIR99021 (GSK3i)</td>
			<td>Stemgent</td>
			<td></td>
			<td>Solvent: DMSO 10mM</td>
		</tr>
		<tr>
			<td>MEK inhibitor PD0325901 (MEKi)</td>
			<td>Stemgent</td>
			<td></td>
			<td>Solvent: DMSO 10mM</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose</td>
			<td>Invitrogen</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline)</td>
			<td>Invitrogen</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (50 mM)</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax ES Cell Qualified Fetal Bovine Serum, 500 ml</td>
			<td>EMD Millipore</td>
			<td>ES-009-B</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax 0.1% Gelatin Solution</td>
			<td>EMD Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G7126-500G</td>
			<td>1.25 M stock conentration in water</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TE (Tris EDTA) pH 8.0 1X</td>
			<td>Quality Biological</td>
			<td>351-011-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF) solution</td>
			<td>Sigma</td>
			<td>93482-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution (20%)</td>
			<td>Quality Biological</td>
			<td>A611-0837-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Q125 sonifier</td>
			<td>Qsonica</td>
			<td>4422</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 solution</td>
			<td>Sigma</td>
			<td>93443-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G for Immunoprecipitation</td>
			<td>Invitrogen</td>
			<td>10004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein A for Immunoprecipitation</td>
			<td>Invitrogen</td>
			<td>10002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein A/Protein G and Magnet Starter Pack</td>
			<td>Invitrogen</td>
			<td>10015D</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>Sigma</td>
			<td>L4408</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads mRNA Purification Kit (for mRNA purification from total RNA preps)</td>
			<td>Invitrogen</td>
			<td>61006</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript Double-Stranded cDNA Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>11917010</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>End-It DNA End-Repair Kit</td>
			<td>Lucigen</td>
			<td>ER81050</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow Fragment (3&#39;&#8594;5&#39; exo-)</td>
			<td>NEB</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr>
			<td>dATP (10 mM)</td>
			<td>Invitrogen</td>
			<td>18252015</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>TrackIt 1 Kb Plus DNA Ladder</td>
			<td>Invitrogen</td>
			<td>10488085</td>
			<td></td>
		</tr>
		<tr>
			<td>E-gel EX agarose gels, 2%</td>
			<td>Invitrogen</td>
			<td>G402002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion high-fidelity 2X master mix with HF buffer</td>
			<td>Thermo Fisher</td>
			<td>F531LPM</td>
			<td></td>
		</tr>
		<tr>
			<td>ChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody</td>
			<td>EMD Millipore</td>
			<td>17-614</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)</td>
			<td>Abcam</td>
			<td>ab32356</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom Cell Culture Plates (6-well)</td>
			<td>Fisher</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Standard Tissue Culture Dishes (10cm)</td>
			<td>Fisher</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor pico</td>
			<td>Diagenode</td>
			<td>B01010001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Flurometer 2.0</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute Reaction Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28204</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28604</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)</td>
			<td>Abcam</td>
			<td>ab9053</td>
			<td></td>
		</tr>
		<tr>
			<td>Labquake rotator</td>
			<td>Thermo Fisher</td>
			<td>400110Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiSeq platform</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO DNase</td>
			<td>Invitrogen</td>
			<td>AM2238</td>
			<td></td>
		</tr>
	</tbody>
</table>

carip-seq and chip-seq: methods to identify chromatin-associated rnas and protein-dna interactions in embryonic stem cells

胚胎干细胞的自我更新和分化受外部信号和转录因子的内在网络、表观遗传调控、combinatorially 对基因组蛋白的转换后修饰的制约。附近基因的表达状态。RNA 也被证明与各种各样的蛋白质交互调控染色质动力学和基因表达。染色质相关 RNA 免疫沉淀 (CARIP) 其次是下一代测序 (CARIP 序列) 是一种新的方法来调查与染色质蛋白相关的 rna, 而染色质免疫沉淀其次是下一代测序 (芯片序列) 是一种功能强大的基因组学技术, 用于在 ES 细胞的全球尺度上绘制出蛋白、转录因子和表观遗传修饰符的平移后修饰位置。在这里, 我们描述了执行 CARIP 和芯片序列的方法, 包括用于下一代测序的库构造, 以生成 ES 细胞中的全球染色质相关 RNA 和 epigenomic 图。

我们通过这个芯片序列协议6成功地审问了 ES 细胞中 H3K4me3、H3K4me2 和 KDM5B 的全基因组绑定。ES 细胞是在无馈线条件下培养的 (图 1), 并如上文所述交叉链接。超声波随后按照芯片序列协议步骤3.2 所述执行, 并通过在2% 琼脂糖凝胶上运行 DNA 进行评估 (图 2)。接下来, 按照步骤4中的描述执行芯片, 并按照步骤6中的?...

Watch this Scientific Journal Video about CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells at JoVE.com

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

在这里, 我们描述了执行芯片序列和 CARIP 的方法, 包括用于下一代测序的库准备, 以生成 ES 细胞中的全球 epigenomic 和染色质相关的 RNA 图谱。

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

carip-seq-chip-seq-methods-to-identify-chromatin-associated-rnas

Research

JoVE Journal

Developmental Biology

9.7K 次观看.  Wayne State University School of Medicine. 在这里, 我们描述了执行芯片序列和 CARIP 的方法, 包括用于下一代测序的库准备, 以生成 ES 细胞中的全球 epigenomic 和染色质相关的 RNA 图谱。

视频: CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的ChIP-SEQ

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

科研

发育生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

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 ...