我们感谢拉斯 Dölken 为初级 T 细胞建立4sU 标记的建议;伊丽莎白. 格拉夫和托马斯 Schwarzmayr 在图书馆代和测序方面提供重要帮助;德克 Eick 和安德鲁弗拉特利提供 RNAPII 和 T 细胞抗体;n. 亨利埃特 Uhlenhaut 和 Franziska Greulich 帮助图书馆准备芯片序列;卡罗琳 c. 弗里德尔得到德意志 Forschungsgemeinschaft (DFG) 赠款 FR2938/7-1 和 CRC 1123 (Z2) 的支持;Elke Glasmacher 由德意志 Forschungsgemeinschaft (DFG) 和德国糖尿病研究中心 (DZD)、亥姆霍兹姆霍兹慕尼黑的赠款 GL 870/1-1 支持。

<ol>
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作者声明他们没有竞争的财政利益。

分析基因调控的整个过程是必要的, 以充分理解细胞适应反应特定的刺激或治疗。结合总 RNA 序列, 4 的 su 序列, 核糖体剖面和芯片序列在不同的时间点导致全面分析的主要过程中的基因调控的时间。对生物过程的深刻理解需要定义实验设置和最佳时间点。
由于研究基因调控的方法迅速改善, 这些协议可以适应快速变化。然而, 它们为研究任何类型细胞的基本基因调控机制提供了最重要的方法。在这里, 我们讨论在使用这些方法时必须考虑的一些缺陷和事实。
单元格:细胞必须是高度可行的, 如果使用主隔离细胞, 必须保证细胞数量的纯度 (例如, 主要 T 细胞的资产管制分析)。即使是轻微的压力细胞也会影响这些非常敏感的测序方法的结果, 并降低新转录或翻译的 RNA 的数量, 并导致在测序结果中不需要的应力响应读数。本协议中提到的离心速度对原 T 细胞进行了优化。因此, 根据细胞类型调整速度。
4sU 对细胞生理学的影响:除了上述的选择, 以验证最小摄动的细胞生理学后, 4sU 增加, 进一步和/或额外的分析可以执行, 特别是当细胞数是有限的。对细胞增殖的影响可以通过简单计数标记和未标记的细胞来验证细胞的加倍时间来测试。核仁应力诱导也可以通过免疫荧光染色 nucleolin 和核的细胞形态学来进行测试。为了进一步验证4sU 的影响, 可以通过将从标记的总 rna 到未标记的总 rna 的读计数关联来测量改变的全球基因表达。
单元格编号:对于体外生成的 T 单元格, 建议至少从Table 2中指示的单元格数量开始。根据单元格类型选择每个方法的适当编号。由于 T 细胞的细胞质和 RNA 比其他细胞少, 很可能是其他细胞数量的减少将是足够的。对于芯片序列, 细胞数高度依赖于所使用的抗体和细胞内感兴趣蛋白质的表达水平。对于组蛋白或 RNAPII 芯片, fewe 细胞可以使用, 而细胞数需要增加时, 转录因子的使用, 特别是如果他们是在低级别表达。
4sU 标记和 RNA biotinylation:使用粘附单元格时, 4sU 标记可以按 Rädle et al所描述的那样定向。19由于单元格非常快速地合并了 4sU, 所以可以直接添加到悬浮液、黏附剂或半黏附细胞的培养基中。
建议用 60-80 µg 的 RNA 开始 biotinylation。然而, 虽然我们没有测试少于30µg, 但可以使用较低数量的 rna. 添加一个 coprecipitant (例如, GlycoBlue), 如果颗粒很难看到, 在沉淀 RNA。达菲et也表明, methylthiosulfonate 活化生物素 (MTS-生物素) 比 HPDP-生物素31更有效地与 4 su 标记 RNA 反应。因此, 也许值得考虑改用 MTS-生物素, 特别是用于恢复小 rna, 后者往往有较少的苷残留 (参照达菲的 biotinylation 协议;参见纯化4个 su 标记的 RNA,实验程序)。
为了恢复新转录的 RNA, 有可能使用顺磁性珠或 RNA 清除珠的选择。总是考虑到这些套件可能或可能不会净化特定 rna。例如, 如果您对 miRNAs 感兴趣, 请考虑使用特定的工具包进行 miRNA 捕获和排序。
对新转录的 RNA 进行量化:要准确量化新转录的 RNA, 测量应由适当的荧光计执行 (请参阅材料表)。在1小时的4sU 曝光, 新转录 rna 代表约 1-4% 的总 RNA。新转录的 1 h 标记的 RNA, 活化 T 细胞由 ~ 90-94% rRNA11组成。
核糖体分析:在建立该方法时, 我们确定使用1.5x 的核酸酶量比原始协议中的建议保证了正确的消化。此外, 没有报告的不良影响, 增加了数量的核酸酶。由于 overdigest 的 RPFs 是相当困难的, 而它们是核糖体蛋白所束缚的 RNA 的一部分, 你仍然可以稍微增加滴定最佳核酸酶消化量。
如果在步进4.4.2 中恢复了不到500的爱国阵线 rna, 重复 rRNA 耗尽和池纯化 RPFs 与 rna 清洁 & Concentrator-5 柱。或者, 在胶体 (步骤 4.5.6) 的 RNA 洗脱过程中, 在凝胶 (步骤 4.5.3) 和池凝胶切片上分别加载两个相同的样本。
我们建议把 RPFs 上的凝胶尽可能紧密地切割到28和 30 nt 波段。这有助于从 rRNAs 和 tRNAs 中消除不需要的片段, 随后将成为库的一部分, 并减少对 RPFs 的排序读取。
还建议在凝胶纯化过程中避免紫外线照射。这会造成 RNA 片段中的缺口, 以及嘧啶脂肪酸, 最终会严重影响到图书馆的准备和测序结果。
库的准备和数据排序:核糖体分析协议能够生成适合于排序的 cDNA 文库。4 su 标签生成的样本可以直接用于图书馆准备与任何适当的 RNA 排序套件。由于新转录的 RNA, 特别是当使用短的标记时间, 可能还没有 polyadenylated, 不应进行聚一选择。相反, 我们强烈建议 rRNA 损耗, 以防止减少实际样品的测序深度。使用 T 细胞, 我们开始与 400 ng 新转录和总 RNA (取决于试剂盒, 见材料), 执行 rRNA 耗尽和减少周期 pcr 扩增, 以减少 pcr 偏倚。图书馆的准备工作可以用较少的起始材料进行。要考虑到库的复杂性, 应优化 PCR 循环的数量。
对于芯片序列, 还有许多工具包可供图书馆准备。在我们的手图书馆准备工作很好从 2 ng 芯片脱氧核糖核酸开始 (参见材料为建议在哪个套件使用)。确保在排序过程中检查颜色平衡的索引。我们建议≥40 x 106的测序深度分别读取4个 su 序列、总 RNA 序列和切屑样本, ≥80 x 106读取核糖体分析样本。测序深度取决于样品和下游生物信息学分析, 应慎重考虑。为了分析 cotranscriptional 拼接的内含子读数, 需要选择 100 bp 配对端测序。
排序偏差:在确定转录、翻译或转录因子结合的全球变化时, 测序已成为黄金标准。近年来, 现有的方法被推向极限, 或者开发出新的技术来测序越来越小的 RNA 起始量。这需要扩增 cDNA, 引入噪声或偏倚。最近, 开发了独特的分子标记 (UMIs), 以实验确定 PCR 引入的重复性。最近, 人们发现, UMIs 只是轻微地提高排序能力和错误发现率的差异基因表达式32。不过, 考虑使用独特的分子标识符 (UMIs) 来控制库的复杂性, 特别是从低数量的 RNA 开始, 以及当需要许多 PCR 周期时。
缓冲区和库存解决方案:所有4个 su 序列和核糖体剖面的缓冲器必须在严格的无 RNase 条件下使用核酸酶水来制备。建议购买预先制作的无核酸酶氯化钠, 三盐酸, EDTA, 柠檬酸钠和水。为确保核酸酶的条件, RNase 净化解决方案可用于清洁吸管或表面。所有芯片序列的缓冲器都需要至少 DNase, 并且可以在室温下存储。在使用和保持冰之前, 总是添加蛋白酶抑制剂, 或者是磷酸酶抑制剂。
生物信息学:分析所有测序数据 (即、芯片序列、RNA 序列和 ribsosome 分析) 涉及质量控制 (例如, 使用 FastQC, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), 适配器修整 (例如, 与cutadapt20), 然后映射到研究中的细胞的参考基因组。对于 rna 序列数据 (总共和4个 su 序列) 以及核糖体分析数据, 需要一个拼接的 RNA 序列映射器, 如 ContextMap 221。对于芯片序列数据 unspliced 对齐, 使用 BWA22是足够的。基因表达式可以使用 RPKM 模型 (每百万片段映射的外显子 Kilobase 读取)1, 在确定每个基因的读计数后使用一个程序,例如featureCounts23来计算。对于来自芯片序列数据的峰值调用, 有许多程序可用,例如、mac24或 GEM25。可以在 R26中执行进一步的下游分析, 特别是使用由 Bioconductor 项目27提供的工具。
在这里, 从核糖体分析中集成 4sU-和总 RNA 水平和平移活动的一个主要挑战是规范化的。解决这个问题的一个经典方法是规范化到保持家庭基因的水平。为了减少由于个体饲养基因随机波动而产生的噪音, 建议不要只使用少量的保留基因, 而是要在较大的集合中中位数,例如由艾森伯格和莱瓦诺编写的 > 3000 家保留基因28.对于从4sU 到总 rna 的比率计算 rna 更替率, 规范化是基于中位更替率 (例如, 假设 rna 半衰期为 5h)29。然而, 由于这不假设对保留基因的整体变化, 我们建议使用独立于规范化的分析方法,例如, 基于关联的不同数据类型的时间序列的聚类, 以确定基因组在激活过程中具有明显的转录和翻译行为。有关不同数据类型的 bioinformatical 集成的详细说明, 请参阅原始发布11。
营业额率和数据集成分析:最近发表的一篇论文33将多路复用基因控制 (MGC) 确定的半衰期与全球方法进行比较, 可以表明半衰期与其他方法 (如) 相比, 与代谢标记法获得的寿命最佳。药物转录的一般抑制)。但是, 应该指出, 半衰期计算之间的差异可能会出现, 并且已被描述为15、34。我们解释了由于长时间4sU 暴露引起的压力反应所引入的大多数问题和差异。因此, 排除 4 su 标记引入的应力响应是必不可少的。为了进一步验证周转率, 我们建议使用 MGCs。
此外, 此处生成的数据集还可用于更综合的数据分析 (例如, 对长的非编码 rna 的调节)35,36。

近年来, rna 测序 (rna 序列) 已成为分析细胞或生物体内所有表达 rna 的标准工具1。然而, 为了了解适应特定刺激/药物的细胞的整个过程, 有必要充分确定所有的基础过程, 从 mRNA 转录到加工、更替和翻译。rna 转录的短期变化几乎不能用总 RNAseq 来衡量, 因为总 rna 的变化取决于因素例如rna 半衰期和转录活动, 这是一个不良的模板, 以反映细胞的适应环境影响2,3。事实上, 已经制定了各种新的测序技术, 允许对基因调节过程中的不同步骤进行分析,4当以正确的方式组合时。该协议描述了如何结合一些相当容易应用的排序技术, 以比较的方式跟踪对 mRNA 的基本层的调节。为了分析转录活动, 我们描述了多种方法, 如基因表达式的 Cap 分析 (笼)5, 本机伸长率转录序列 (净序)6, 以及全基因组的核运行 (的序列)7,8, 以及 bromouridine 序列 (老兄) 和 4-thiouridine 序列 (4 su), 它使用在新转录的 RNA9,10中合并的代谢产物, 仅举几个。当笼子识别准确的转录起始点时, 网络序列和增加序列提供了关于阅读方向的更准确的信息, 4 的 su 序列 (这里描述的方法) 只检测新转录的 RNA。然而, 4 的 su 序列是高度敏感的, 可以应用于不同的时间框架来测量积极变化细胞中的定量转录活性, 以及 mRNA 处理的数量变化 (在几分钟内发生)9, 11,12。此外, 4 su 是理想的结合 rna 序列计算 rna 更替率的基因9。mRNA 的转录是由 RNA 聚合酶 II (RNAPII) 进行的, 这反过来受到多种因素的影响, 如转录因子、组蛋白修饰和一般的激活剂/阻遏, 甚至可以是转录的一部分。复杂.为了测试有多少基因/启动子/增强器区域受一个因素的约束, 芯片序列已被开发, 这是现在的标准方法为此目的, 由于许多商业上可用的抗体13。但是, 尽管 ChIPseq 提供了有关调节因素绑定的明确信息, 但它并不反映它是否确实导致了转录14的变化。因此, 用4的 su 序列进行芯片序列是这类生物问题的理想组合。基因表达调控也可能在以后的阶段发生, 因为 mRNA 和蛋白质水平不一定关联15,16, 表明有可能对平移或后转化的重要调节级别, 具体取决于上下文。在 2011, 核糖体的分析首先与 RNA 序列结合, 现在是选择的方法来量化迅速发生的蛋白质变化, 因为仍然有一些敏感性限制与质谱17。事实上, 从这些方法获得的笔译率已证明对蛋白质水平的变化 (至少是长期变化的衡量) 提供了相对较好的估计, 并允许对翻译过程有更详细的看法,例如确定起始端和可选翻译框架17。所有四方法的组合使用可以在稳定状态、不同单元格类型之间或在快速变化的单元格11的时间序列实验中使用。这一用途提供了一个全基因组范围内的变化转录因子结合影响 RNA 转录, 处理和翻译。

<table>
	<tbody>
		<tr>
			<td>4sU-labeling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-thiouridine (100 mg)</td>
			<td>Carbosynth</td>
			<td>13957-31-8</td>
			<td>Prepare 50 mM stock in sterile H2O/PBS; store at &#8211;20&#176;C in aliquots of 50-500 &#181;l; do not refreeze.</td>
		</tr>
		<tr>
			<td>1.5 ml safe-lock tubes</td>
			<td>Eppendorf</td>
			<td>30121589</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>1.5 ml screw-top polypropylene tubes</td>
			<td>Sarstedt</td>
			<td>72692005</td>
			<td>Compatible with Dimethylformamide</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td>Compatible with Dimethylformamide</td>
		</tr>
		<tr>
			<td>2.0 ml screw-top polypropylene tubes</td>
			<td>Sarstedt</td>
			<td>72694005</td>
			<td>Compatible with Dimethylformamide</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>Compatible with Dimethylformamide</td>
		</tr>
		<tr>
			<td>Agencourt RNAClean XP</td>
			<td>Beckman Coulter</td>
			<td>A63987</td>
			<td>We recommend to use these paramagnetic beads. Aliquot and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma Aldirch</td>
			<td>372978</td>
			<td>WARNING &#8211; HAZARDOUS TO HEALTH</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Roth</td>
			<td>6908.1</td>
			<td>Prepare as 100 mM DTT in nuclease-free H2O; always prepare fresh</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>1.00983.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link Biotin-HPDP (50 mg)</td>
			<td>Pierce</td>
			<td>21341</td>
			<td>Prepare 1 mg/ml stock solution by dissolving 50 mg Biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4&#176;C in aliquots of 1 ml. DMF dissolves some plastic materials. We recommend to use glass pipettes to transfer DMF from ist stock glass bottle to 50 ml Falcon tubes.</td>
		</tr>
		<tr>
			<td>High Sensitivity DNA Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>1.09634.1011</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (5M)</td>
			<td>Sigma Aldrich</td>
			<td>71386</td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>nuclease-free EDTA (500 mM ), pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>Nuclease-free H2O</td>
			<td>Sigma Aldrich</td>
			<td>W4502</td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>nuclease-free Tris Cl (1M), pH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td>Stock solution</td>
		</tr>
		<tr>
			<td>Phase Lock Gel Heavy tubes (2.0 ml)</td>
			<td>5Prime</td>
			<td>2302830</td>
			<td>Use in step 1.3.4.</td>
		</tr>
		<tr>
			<td>Polypropylene 15&#160;ml centrifuge tubes</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>Or equivalent; they have to tolerate up to 15,000 &#215; g</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent (200 ml)</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Use this or equivalent TRI reagent for RNA isolation, WARNING &#8211; CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol)</td>
		</tr>
		<tr>
			<td>Qubit RNA HS assay kit</td>
			<td>Life Technologies</td>
			<td>Q32852</td>
			<td>Use this kit for quantifying RNA quantity in step 1.4.11</td>
		</tr>
		<tr>
			<td>RNeasy MinElute Kit</td>
			<td>Qiagen</td>
			<td>74204</td>
			<td>Optional; includes Buffer RLT</td>
		</tr>
		<tr>
			<td>Sodium citrat</td>
			<td>Sigma Aldrich</td>
			<td>C8532</td>
			<td>Prepare 1.6 M stock solution using nuclease-free water</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;Macs Streptavidin Kit</td>
			<td>Miltenyi</td>
			<td>130-074-101</td>
			<td>Store at 4&#176;C, includes &#181;Macs columns used in step 1.4.6. (store at RT)</td>
		</tr>
		<tr>
			<td>Cell viability and stress assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Annexin V Apoptosis Detection Kit I</td>
			<td>BD Biosciences</td>
			<td>559763</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Sigma-Aldrich</td>
			<td>T9033</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>p53</td>
			<td>abcam</td>
			<td>ab26</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>p-EIF2a (Ser51)</td>
			<td>Cell Signaling</td>
			<td>9721</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>BH3I-1</td>
			<td>Sigma-Aldrich</td>
			<td>B 8809</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4sU Washing Buffer</td>
			<td></td>
			<td>store at RT</td>
			<td>100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O</td>
		</tr>
		<tr>
			<td>Biotinylation Buffer (10x)</td>
			<td></td>
			<td>store at 4 &#176;C</td>
			<td>100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water; make aliquots of 1 ml; store at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNA precipitation buffer</td>
			<td></td>
			<td>store at RT</td>
			<td>1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer instrument</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Nano Kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td>Use this kit to verify RNA integrity in step 1.3.10</td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>UV/VIS spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 1000</td>
			<td>Or equivalent. Use in step 1.2.2/3.1.8/3.3.3/3.4.3</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Thermo Scientific</td>
			<td>Heraeus Multifuge X3R</td>
			<td rowspan="3">Or equivalent equipment capable of reaching 13,000&#160;&#215;&#160;g</td>
		</tr>
		<tr>
			<td>High-speed rotor</td>
			<td>Thermo Scientific</td>
			<td>Fiberlite F15-6 x 100y</td>
		</tr>
		<tr>
			<td>Adaptors for 15 ml tubes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated table-top centrifuge</td>
			<td>Eppendorf</td>
			<td>5430 R</td>
			<td>Or equivalent.</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>Thermomixer C</td>
			<td>Or equivalent.</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td>One stand holds 8 &#181;Macs columns.</td>
		</tr>
		<tr>
			<td>Ultra-fine scale</td>
			<td>Mettler Toledo</td>
			<td>ML204T</td>
			<td>Or equivalent.</td>
		</tr>
		<tr>
			<td>E-Gel iBase Power System</td>
			<td>Invitrogen</td>
			<td>G6400UK</td>
			<td>For RNA gels; or equivalent.</td>
		</tr>
		<tr>
			<td>E-Gel EX 1% agarose precast gels</td>
			<td>Invitrogen</td>
			<td>G4020-01</td>
			<td>For RNA gels; or equivalent.</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet-1 each</td>
			<td>Life Technologies</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Sigma</td>
			<td>R2020</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>TruSeq stranded total RNA library prep kit</td>
			<td>Illumina</td>
			<td>RS-122-2201</td>
			<td>Or equivalent. For T cells we used 400 ng 4sU and Total RNA with 11 cycles for PCR amplification. rRNA depletion is included in this kit</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>use a Nanodrop or equivalent instrument to measure RNA concentration</td>
		</tr>
		<tr>
			<td>Ribosome Profiling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Ribo Profile kit (Mammalian or Yeast)</td>
			<td>Illumina</td>
			<td>RPYSC12116 (Yeast)</td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Ribo Profile kit (Mammalian or Yeast)</td>
			<td>Illumina</td>
			<td>RPHMR12126 (Mammalian)</td>
			<td></td>
		</tr>
		<tr>
			<td>Illustra MicroSpin S-400 HR Columns</td>
			<td>GE Healthcare</td>
			<td>27-5140-01</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator-25 kit</td>
			<td>Zymo Research</td>
			<td>R1017</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator-5 kit</td>
			<td>Zymo Research</td>
			<td>R1015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat)</td>
			<td>Illumina</td>
			<td>MRZG126 or MRZG12324</td>
			<td></td>
		</tr>
		<tr>
			<td>(High Sensitivity DNA Kit)</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td>Already needed for 4sU-seq</td>
		</tr>
		<tr>
			<td>All other consumables and equipment are listed in the User guide</td>
			<td>!!!</td>
			<td></td>
			<td>Carefully read the user guide and order required consumables in advance (consider a long delivery time for some consumables e.g. gels)</td>
		</tr>
		<tr>
			<td>ChIP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris-HCl (pH 8.0)</td>
			<td>gereral lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp Plus Marker</td>
			<td>Thermo Fisher</td>
			<td>SM0323</td>
			<td></td>
		</tr>
		<tr>
			<td>16 % Formaldehyde</td>
			<td>Thermo Fisher</td>
			<td>28908</td>
			<td>Add to a final concentration of 1 %</td>
		</tr>
		<tr>
			<td>70% EtOH</td>
			<td>gereral lab supplier</td>
			<td></td>
			<td>Always prepare fresh</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>gereral lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agencourt RNAClean XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63987</td>
			<td>We recommend to use these paramagnetic beads. Aliquot and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>ChIP library preparation kit</td>
			<td>KapaBiosystems</td>
			<td>KK8504</td>
			<td>Or use the kit of your choice</td>
		</tr>
		<tr>
			<td>DNA low bind microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>Z666548-250EA</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10004D</td>
			<td>Use these superparamagnetic beads coupled to protein G in step 4.3.1.; Bring to RT before use</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>gereral lab supplier</td>
			<td></td>
			<td>Prepare a 2M stock solution</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>10-901-393-001</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>Use this kit (or equivalent) to purify chromatin in step 4.2.4.</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor (PhosStop)</td>
			<td>Roche</td>
			<td>4906837001</td>
			<td>Add freshly to the buffer and keep on ice</td>
		</tr>
		<tr>
			<td>Power SYBRgreen Master mix</td>
			<td>Thermo Fisher</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor (cOmplete, EDTA-free)</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Add freshly to the buffer and keep on ice</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Invitrogen</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay kit</td>
			<td>Invitrogen</td>
			<td>Q32851</td>
			<td>Use this kit for quantifying DNA quantity in step 4.6.4. on a Qubit Fluorometer</td>
		</tr>
		<tr>
			<td>Rnase, DNase free</td>
			<td>Roche</td>
			<td>11-119915001</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon sperm (sonicated to around 100bp)</td>
			<td>Sigma</td>
			<td>D1626</td>
			<td></td>
		</tr>
		<tr>
			<td>TE pH 8.0</td>
			<td>gereral lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies (ChIP grade if possible)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-RNA Pol II [8WG16]</td>
			<td>abcam</td>
			<td>ab817</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Histon H3K36me3</td>
			<td>abcam</td>
			<td>ab9050</td>
			<td></td>
		</tr>
		<tr>
			<td>or antibody of interest</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binding/Blocking buffer</td>
			<td></td>
			<td>Store at RT</td>
			<td>PBS with 0.5 % BSA and 0.5 % Tween 20</td>
		</tr>
		<tr>
			<td>Cell-Lysis buffer</td>
			<td></td>
			<td>Store at RT</td>
			<td>5 mM Pipes [pH 8.0], 85 mM KCl, and 0.5 % NP40</td>
		</tr>
		<tr>
			<td>ChIP IP buffer</td>
			<td></td>
			<td>Store at RT</td>
			<td>0.01 % SDS; 1. 1% Triton X-100;1.2&#160;mM EDTA; 16.7&#160;mM Tris-HCl, pH 8.1; 16.7&#160;mM NaCl</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td></td>
			<td>Store at RT up to 6 months</td>
			<td>10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 300 mM NaCl and 0.5 % SDS</td>
		</tr>
		<tr>
			<td>Nuclei-Lysis buffer</td>
			<td></td>
			<td>Store at RT</td>
			<td>50 mM Tris [pH 8.0], 10 mM EDTA, and 1 % SDS</td>
		</tr>
		<tr>
			<td>Wash buffer I</td>
			<td></td>
			<td>Store at RT</td>
			<td>0.1 % SDS; 1 % Triton X-100; 2 mM EDTA; 20mM Tris-HCL pH 8.1; 150 mM NaCl</td>
		</tr>
		<tr>
			<td>Wash buffer II</td>
			<td></td>
			<td>Store at RT</td>
			<td>0.1 % SDS; 1 % Triton X-100; 2&#160;mM EDTA; 20&#160;mM Tris-HCL pH 8.1; 500&#160;mM NaCl</td>
		</tr>
		<tr>
			<td>Wash buffer III</td>
			<td></td>
			<td>Store at RT</td>
			<td>0.25&#160;M LiCl; 1% NP-40; 1&#160;mM EDTA; 10&#160;mM Tris-HCl, pH 8.1</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer instrument</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td>use this instrument for electrophoretical analysis</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor TBX microtubes 1.5 ml</td>
			<td>Diagenode</td>
			<td>C30010010</td>
			<td></td>
		</tr>
		<tr>
			<td>or tubes special for your sonication device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor sonication device or sonication device of your choice</td>
			<td></td>
			<td></td>
			<td>Sonication of T cells with Bioruptor: 20 - 25 cycles (30 s on, 30 s off at high in two 1.5 ml bioruptor microtubes with 500 &#181;l each tube)</td>
		</tr>
		<tr>
			<td>Magnetic stand for tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Use this Fluorometer for quantifying low amounts of RNA/DNA</td>
		</tr>
	</tbody>
</table>

real-time analysis of transcription factor binding, transcription, translation, and turnover to display global events during cellular activation

激活后, 细胞迅速改变其功能程序, 从而, 他们的基因表达谱。基因表达的巨大变化, 例如, 在细胞分化, 形态发生, 功能刺激 (如激活免疫细胞), 或在接触到药物和其他因素的地方环境。根据刺激和细胞类型, 这些变化迅速发生, 并在任何可能的基因调控水平。将应答细胞的所有分子过程显示给某种刺激/药物是分子生物学中最困难的任务之一。在这里, 我们描述了一个协议, 可以同时分析多层次的基因调控。我们比较, 特别是转录因子结合 (染色质-免疫沉淀排序 (芯片序列)), de从头转录 (4-thiouridine 序列 (4 su)), mRNA 处理和营业额以及翻译 (核糖体分析)。通过结合这些方法, 可以显示一个详细的和全基因组范围的行动过程。
在分析快速适应或改变系统时, 特别推荐测序新转录 RNA, 因为这描述了4sU 暴露期间所有基因的转录活动 (不管它们是 downregulated 的还是不确定的)。总 rna 序列和核糖体分析的组合使用另外允许计算 rna 营业额和翻译率。对高通量测序结果的生物信息学分析, 可以为数据的分析和解释提供多种方法。生成的数据还可以跟踪联合转录和替代拼接, 只是提到一些可能的结果。
这里描述的组合方法可以适用于不同的模型有机体或细胞类型, 包括主要细胞。此外, 我们为每个使用的方法提供详细的协议, 包括质量控制, 并讨论潜在的问题和隐患。

4sU 标记: 验证最佳4sU 标记条件 (凋亡、核应力、胞浆应力)、时间和浓度:高水平的4sU 可以抑制 rRNA 的生产和加工, 诱导细胞质以及核应力30。因此, 需要对细胞的4诱导应力和细胞凋亡进行检测。建议采用西方印迹分析方法可视化 p53 积累, 表明核应力, 增加 phospho-EIF2a 水平, 显示胞浆应力和荧光活化细胞分类 () 对细胞凋亡的分析。高浓度和长时间接触4sU 或药物, 如 thapsigargin 或亚砷酸可用于诱发细胞压力。为诱导凋亡或细胞死亡, 细胞以 BH3I-1 (500 ng/µL) 治疗, 或在95摄氏度 (热休克) 中孵化5分钟。蛋白 V/7-AAD 染色用于测定凋亡 (蛋白 V) 和死亡 (7) 细胞。标记体外生成的主 Th1 细胞为0.5 小时, 500 µM 4sU (最终浓度) 或 1 h, 200 µM 4sU 既不诱发细胞压力或凋亡的迹象 (图 1), 但导致足够的4sU 合并。
RNA 标记时间也可以缩短 (≤5分钟), 这导致短寿命的内含子序列的增加比更长的标记时间。为了可视化共转录拼接率, 4 su 标记时间不应超过30分钟。有关4sUlabeling 的详细信息, 请参阅 Rädle et。19
质量控制:rna 完整性在处理 rna 时是非常重要的。通过 electrophoretical 分析检查4个 biotinylation 标记 rna 的 rna 质量是最方便的 (参见材料表)。考虑从步骤2.2.2 中验证分离的 rna, 特别是在使用它进行总 RNA 序列排序时。rna 完整性编号 (≥8) 应被确认, 以确保 rna 完整性, 以便进一步处理 (图 3)。
Electrophoretical 分析也可以用来验证新转录的 RNA。要知道, 新转录的 rna 含有明显较不成熟的 rRNAs 与总 RNA 相比, 典型的 rRNA 带是不太突出。
核糖体分析: cdna 文库的 PCR 扩增: cdna 放大 (步骤 4.9.1) 是确保良好测序结果的关键步骤。通过 electrophoretical 分析对放大后的库进行分析。一个好的放大库示例显示了大约 140-160 bp (图 4A) 的峰值。应避免使用过多的适配器脂肪酸 (图 4B), 并应根据制造商的协议 (PCR 产品的页面纯化) 进一步纯化这些样品。过多的模板或太多的 PCR 周期会导致过度放大, 其特征是高于预期的分子量带、涂抹的 PCR 产品和适配器二聚体产品 (图 4C)。对于大多数样本 1-5 µL 的发送 cDNA 和 9 PCR 周期的扩增, 通常会产生足够数量的正确的 PCR 产品。
芯片: 染色质剪切:每种类型的细胞都需要调整最佳的剪切条件。预先确定剪切条件 (例如、周期数、高或低功耗)。使用相同数量的细胞和相同体积的测试目的, 因为较低的细胞密度增加剪切效率。尽量避免过度或不受剪切的染色质。大的染色质碎片可以通过堵塞来显著地影响芯片的结果, 过度剪切可以破坏兴趣蛋白上的表位, 导致抗体的结合效率降低。在本实验中, 当剪切染色质的主要分数约为 1000 bp 或稍低 (图 5A) 时, 取得了最佳效果。
通过 qPCR 验证芯片:在启动芯片之前, 最好测试使用的抗体是否适合芯片 (如果可能的话, 使用芯片级抗体) 的芯片 qPCR。在启动库准备之前, 验证芯片是否按 qPCR 排序 (请参见步骤 5.6.5)。设计引物, 绑定到已知的兴趣蛋白质的目标站点。如果一个基因中的确切靶点是未知的, 几个引物对可以用来扫描基因和相关的调控元素。对于 Th1 单元格的 RNAPII 芯片Ifng, 该在刺激时转录上调, 肌动蛋白引物可用作正向控制。Sox9和胰岛素作为一个负控制, 因为这些基因不是在 Th1 细胞中表达的 (图 5B)。记住不要使用外显子-跨越引物, 这通常用于 qPCR 的 mRNA。IgG 控制也可用于证明所用抗体的特异性。Immunoprecipitated DNA 可以用合适的荧光计来测量 (参见材料表)。免疫球蛋白控制的 nonspecifically 绑定 dna 的数量应该大大降低, 与 DNA 的数量绑定的抗体的利益。
复制: 生物学意义的证明:强烈建议对从同一池中开始的所有方法执行动力学实验, 以确保单元格对所有未经处理和治疗的样本具有相同的标识 (图 2)。然而, 建议采取小整除数的主要时间点为每个方法比较样本与生物复制 (例如, 通过 qPCR, 资产管制分析).这允许粗略估计, 如果两个复制的治疗是可重现的, 你可以进行排序。复制的验证应通过 bioinformatical 分析进行。结果的重现性可以用散布图 (图 6) 来评估复制和可视化之间的 FPKM 值之间的相关性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56752/56752fig1.jpg"> 图 1: 验证无扰动细胞生理学的最佳 4 su 标记条件 (从 Davari et al.11) (A) 通过资产管制分析法检测细胞凋亡:体外生成的 Th0 细胞以不同浓度的 4sU (括号内表示) 分别治疗0.5 小时、1小时和2小时。BH3I-1 治疗诱导蛋白 V 的凋亡, 而热休克 (5 分钟95摄氏度) 被用来诱导细胞死亡, 由7。(B) 对4sU 治疗和活化 T 细胞 p53 的西方印迹分析: 样品被标记为200µM 4sU, 显示激活时间, 但标记为500µM 4sU 的 0.5 h 时间点除外。(C) 在具有相同标记条件 (B) 的激活 Th1 细胞中 phospho-EIF2a 和总 EIF2a 的西方印迹分析。Thapsigargin 被用来作为一个积极的控制。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56752/56752fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56752/56752fig2.jpg"> 图 2: 用于跟踪全基因组变化的动力学设置示意图 该方案说明了将4的 su 序列、总 RNA 序列、核糖体剖面和芯片序结合在细胞治疗上研究全基因组变化的设置。池单元格, 并预留所需的单元格数以进行未经处理的控制。为每个方法和时间点处理剩余的单元格和拆分。标签未经处理的/治疗的细胞为4苏序列与4sU 如所述。每个方法的时间点和样本取决于所检查的特定生物学问题。为每个时间点和方法抽取样本, 并遵循协议的专用部分。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56752/56752fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56752/56752fig3.jpg"> 图 3: 4 个 su 标记 RNA 的质量控制 Bioanalyzer 对活化 Th1 细胞的总 rna 和生物素化 rna 进行了分析。18S rRNA 和 28S rRNA 显示, 并且 rna 完整性数字 (玲) 由仪器给确定 rna 的完整性。应≥8, 以确保 RNA 的完整性。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56752/56752fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56752/56752fig4.jpg"> 图 4: 核糖体分析库的 Bioanalyzer 配置文件 (A) 一个好的库: 该示例显示了预期大小范围 (140-160 bp) 的峰值, 不需要进一步纯化。(B) 本示例显示了相对于所需产品 (140-160 bp) 的过多适配器二聚体放大产品 (120 bp)。这个图书馆需要进一步的净化。(C) 过度放大的样本: 高于预期的分子量峰值和涂抹的 PCR amplicons 是可见的 (由箭头表示)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56752/56752fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56752/56752fig5.jpg"> 图 5: qPCR 剪切和验证芯片后的最佳染色质尺寸 (A) 琼脂糖凝胶图片显示被剪切的染色质的最佳片段大小从三个样品被剪切了25个周期在 sonicator 和纯化如在协议之前描述。(B) 总 RNAPII 芯片 (反 RNA II、8WG16、ab817) 的 Q PCR 结果表示为输入的百分比。Ifng和肌动蛋白引物用作阳性, 而Sox9和胰岛素为负控制 (两种基因均未在活化 Th1 细胞中表达)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56752/56752fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56752/56752fig6.jpg"> 图 6: 生物复制的比较 (来自 Davari et ) 的数字。11) 在激活 Th1 细胞刺激后, 新转录 (4sU) RNA 4 h 复制之间的表达值 (FPKM) 的代表性散点图比较。绿线表示相等的 FPKM 值和秩相关在每个情节被表明。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>标签持续时间 (分钟)</td>
			<td>推荐的4sU 浓度 (µM)</td>
		</tr>
<tr>
<td>120</td>
			<td>100-200</td>
		</tr>
<tr>
<td>60</td>
			<td>200-500</td>
		</tr>
<tr>
<td>15-30</td>
			<td>500-1000</td>
		</tr>
<tr>
<td>< 10</td>
			<td>500-2000</td>
		</tr>
</tbody></table>表 1: 推荐的4sU 浓度 (从 Rädle, et al.19) 推荐的4sU 浓度的范围是指不同的标签时间。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>方法</td>
			<td>单元格编号</td>
			<td>RNA 数量</td>
		</tr>
<tr>
<td>4 su 标记</td>
			<td>≥2 x 107
</td>
			<td>≥60µg</td>
		</tr>
<tr>
<td>核糖体分析</td>
			<td>≥2 x107
</td>
			<td></td>
		</tr>
<tr>
<td>芯片序列</td>
			<td>≥2 x 107 -3 x 107
</td>
			<td></td>
		</tr>
</tbody></table>表 2: 所需的初级 T 细胞数量 每个方法所需的主要 T 单元格的最小数量。使用其他单元格类型时, 金额可以减少。

Watch this Scientific Journal Video about Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation at JoVE.com

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

该协议描述了芯片序列的组合使用, 4 的 su 序列, 总 RNA 序列, 和核糖体分析的细胞系和主细胞。它能够跟踪转录因子绑定、从头转录、RNA 处理、营业额和翻译的变化, 并在激活和/或快速变化的细胞中显示事件的总体过程。

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

real-time-analysis-transcription-factor-binding-transcription

Nanyang Technological University

Research

JoVE Journal

Genetics

13.3K 次观看.  Helmholtz Zentrum München. 该协议描述了芯片序列的组合使用, 4 的 su 序列, 总 RNA 序列, 和核糖体分析的细胞系和主细胞。它能够跟踪转录因子绑定、从头转录、RNA 处理、营业额和翻译的变化, 并在激活和/或快速变化的细胞中显示事件的总体过程。

视频: Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

使用BrUTP链特异性转录运行的（TRO）方法终止转录分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...