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>

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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>10&lt;sup&gt;6 &lt;/sup&gt;or 10&lt;sup&gt;7 &lt;/sup&gt;units</td></tr><tr><td>GSK3&amp;beta; 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&#039;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&#039;&amp;rarr;5&#039; 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

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

遗传学

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

生物学

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

神经科学

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

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

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染色质监管机构和各国的全基因组快照

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射