作者希望感谢稀有疾病模型和机制 (RDMM) 网络 (由加拿大卫生研究所 (卫生研究院) 和加拿大基因组资助)。CK 获得 UROP 奖学金 (渥太华大学) 的支持。DLJ 得到加拿大 Vanier 研究生奖学金的支持。由加拿大卫生研究院 (卫生研究院) 博士后奖学金奖资助。作者感谢美国遗传学学会批准发布这项协议, 并使用修改的补充图5的修订,等等。

有关动物的程序已获批准, 并符合加拿大动物保育委员会和渥太华大学动物保育委员会提供的动物保育指引。
1. 准备
<ol><li>通过用高压釜胶带在其内部表面上贴上9厘米的培养皿盖, 准备解剖表面。在立体声显微镜下定位。</li>
	<li>放置 3-5 天后受精 (dpf) 斑马鱼幼虫在培养皿和麻醉˜1.5 毫米 Tricaine 1x E3 胚介质 (5 毫米氯化钠, 0.17 毫米氯化钾, 0.33 毫米 CaCl2, 0.33 毫米 MgSO4, pH 7.2)。</li>
	<li>用剪刀或剃刀刀片切断 P1000 吸管尖端的末端, 直径为2毫米, 以容纳3的 dpf 幼虫, 压力最小。</li>
</ol>2. 斑马鱼幼虫的鳍片修剪
<ol><li>使用改良的 P1000 微提取麻醉幼虫, 将斑马鱼幼虫放在培养皿盖上放置的高压釜胶带上。</li>
	<li>用微去除幼虫周围的过量 Tricaine 溶液。该区域应尽可能干燥, 以成功的部分, 并拿起鳍, 但仍然湿, 以确保生存。</li>
	<li>使用微手术刀, 在一个立体声显微镜下的尾鳍切片, 如图 1所示, 如威尔金森et al16。在切口期间, 在尾翅的颜料间隙部位, 在血液循环的极限处应用稳定的下行压力 (图 1A)。施加温和的压力以避免损坏脊索。</li>
	<li>在显微镜下可视化切片鳍, 并将该片放置在 microscalpel 刀片尖端的顶端 (图 2A)。将含有鳍的 microscalpel 放在一小片滤纸的表面上。 注意: 由于在横断组织中存在黑色素细胞, 这一步骤允许将鳍可视化为一个小黑点 (图 2B)。</li>
	<li>使用剪刀和镊子 (图 2C), 将含有翅片的滤纸转移到一个96井 PCR 板上, 其中包含 25 ul 每井50毫米氢氧化钠溶液 (图 2D)。</li>
	<li>准备一个平底的96井板, 根据 PCR 板的标签对油井进行编号。使用改良的 P1000 吸管填充200µL 新鲜 1x E3 胚培养基, 小心收集斑马鱼幼虫使用温和的压力。将幼虫放在96井组织培养板中, 与 PCR 板相同的井数 (图 2E)。</li>
	<li>确保过滤器的纸张在溶液中被充分淹没 (图 2F)。清洁 microscapel 和镊子之间的刀片, 以防止交叉污染, 通过浸渍到70% 乙醇解决方案。用干净的纸巾擦拭刀片/擦拭。</li>
	<li>重复步骤 2.1-2.7, 直到所有的胚胎被修剪。</li>
	<li>将幼虫储存在96井板中28摄氏度, 直到鉴定出基因型。 注意: 胚胎介质不需要改变, 因为该协议可以很容易地完成在不到2天。如果需要更多时间, 建议进行媒体更改。</li>
</ol>3. 基因组 DNA 提取
<ol><li>密封96井 PCR 板和离心机的样品在 1000 x g 1 分钟, 以确保所有的过滤器纸被淹没在氢氧化钠溶液中。</li>
	<li>为组织裂解, 加热样品在一个 thermocycler 在95°c 为5分钟然后冷却到4°c 为10分钟。</li>
	<li>添加 6 ul 500 毫米三 HCl, pH 8.0 到每个样品。涡。</li>
	<li>在室温下, 将该板材离心为 1500 x g, 5 分钟。</li>
	<li>每个 PCR 反应使用 1.5 ul 的 DNA 上清。</li>
	<li>准备 PCR 的基因型的幼虫, 如表 1-2所示。 注: 本协议测试了两个应用: 多重 PCR 反应揭示的基因型使用琼脂糖凝胶9, 和 heteroduplex 熔融试验 (HMA) 揭示基因型的聚丙烯酰胺凝胶电泳 (页)17。</li>
</ol>4. 用螯合树脂提取替代基因组
<ol><li>完成步骤2.5 通过添加带修剪翅片的滤纸到96井 PCR 板, 其中含有 30 ul 5% 螯合树脂 (含配对 iminodiacetate 离子的苯乙烯-二乙烯苯共聚物)。 注: 这种树脂通常用于组织裂解和制备 PCR 准备好的 DNA 快速有效的方式18。</li>
	<li>按照指示执行步骤 2.6-2.8。</li>
	<li>密封96井 PCR 板, 并简要离心样品, 以确保所有过滤纸被淹没在螯合树脂内。</li>
	<li>为组织裂解, 加热样品在一个 thermocycler 在95°c 为15分钟然后冷却到4°c 为10分钟。 注意: 这一步允许在 DNA 和 RNA 保持溶液时, 将极性树脂珠子的溶解和随后的结合到细胞成分中。</li>
	<li>简要地离心样品颗粒树脂珠并且获取脱氧核糖核酸在悬浮。</li>
	<li>按照步骤 3.5-3.6 完成基因分型程序。</li>
</ol>5. 应用 1: 多重 PCR 后琼脂糖凝胶分析
<ol><li>下表 1, 建立了多重 PCR 反应, 以使纯合突变体, 杂合子和野生幼虫之间的区别。利用表 3中描述的循环条件, 使用96井热循环仪进行 PCR 反应。 注: pcr 反应也可以在任何其他常规 pcr 热循环仪进行。这个例子描述了使用的 PCR 策略. 9 ) 在同一复用反应中识别aldh7a1和 5-bp 插入突变等位基因。</li>
	<li>在硼酸钠缓冲液中制备1% 琼脂糖凝胶, 染色 1x GelRed。 注: 20x 硼酸钠缓冲液由40毫升10米氢氧化钠制成, 1800 毫升 ddH2O, pH 调整为 8.5, 硼酸76克, 然后用 ddH2O 完成2升最终容积。</li>
	<li>在1x 硼酸钠缓冲液中运行凝胶15分钟, 250 v。为方便这一过程, 如果可能的话, 使用与多通道滴管兼容的凝胶系统, 通过减少加载样本的时间来实现更高的吞吐量能力。</li>
	<li>图像的凝胶下紫外线 (UV) 光允许歧视的不同基因型。</li>
</ol>6. 应用 2: Heteroduplex 熔融试验
<ol><li>准备12% 页凝胶使用1.5 毫米间隔板和15样品梳。用12.21 毫升的 ddH2O、2.52 毫升10x 三/硼酸/EDTA ()、10毫升30% 丙烯酰胺、250硫酸铵 (APS) 和 10% tetramethylethylenediamine (TEMED) 的 ul 来制备两页凝胶。使用1x 缓冲器 (0.089 米三盐酸, 0.089 米硼酸和0.002 米乙二胺乙酸 (EDTA)) 作为运行缓冲器。</li>
	<li>将 PCR 产品加热94摄氏度, 5 分钟。</li>
	<li>冷却管在冰或在 thermocycler 10 分钟在4°c。</li>
	<li>每 PCR 样品负载 10 ul 和8个分子量标记 ul。</li>
	<li>使用垂直电泳系统1小时, 或直到分子量标记几乎达到玻璃板的脚线, 以1x 的150伏运行该页面。</li>
	<li>图像的凝胶在紫外线下, 以允许歧视的不同基因型。</li>
</ol>

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作者声明他们没有竞争的财政利益。

基因编辑工具已经被用来精确地引入突变和转基因在过去几年斑马鱼5,6,7,8。在斑马鱼进行疾病建模时, 早期幼虫或幼年表型通常观察到9、12、13、14。这里提出的协议描述了从小斑马鱼幼虫尾活检中提取 PC...

斑马鱼 (斑马斑马)是一种脊椎动物有机体, 广泛用作疾病调查的模型, 以及治疗假说1,2,3的临床前检验。这些动物有短的世代时间, 容易处理, 显示高繁殖率, 低费用, 并且容易地接受基因操作通过显微注射 DNA 在胚胎4。通过新的转基因和基因编辑技术的不断发展, 如锌指核酸 (ZFN)5, 核酸 (TALENs)6,7和聚集定期 Interspaced 短回文重复 (CRISPR)/CRISPR 相关 9 (Cas9) 系统8, 斑马鱼准备大大提高对几个病理条件的理解。这些技术已经被用来在斑马鱼的体细胞和生殖细胞中产生靶向性的挖空, 有效地培养转基因动物8。最近在文献中的例子包括成功地开发了癫痫的遗传模型和代谢紊乱在斑马鱼3,9,10,11,12.
随着斑马鱼基因组编辑的进展, 快速而可靠的基因分型已成为不可否认的限速步骤。在预测基因组编辑目标站点附近的特定 DNA 突变的识别是该协议的一个重要阶段。传统上, 鳍片修剪的基因分型技术通常只在青少年或成年斑马鱼身上进行。然而, 将斑马鱼饲养到成年 (> 2 月) 的时间大大延缓了研究的进展。在许多情况下, 由于关键基因的挖空 (例如疾病建模) 或功能突变导致的表型效应, 成年无法实现。此外, 一些化验需要收集幼虫, 如在 RNA 或代谢物提取, 从而涉及事先鉴定的基因型, 这可能是挑战时, 只有特殊的基因组技术可用。上述例子突出了幼虫基因分型技术的重要性, 同时也是精确的, 能够充分恢复和正常发育的幼虫。早期斑马鱼的基因分型目前似乎没有得到广泛应用;最近的一些疾病模型的出版物反映了这一点, 其中幼虫基因型是通过验尸后基因类型确定的, 而不是在实验12、13、14的执行之前分基因。本文提出了一种幼虫夹鳍策略, 旨在改进以前建立的基因分型程序。
过去, 采用蛋白酶 K 消解基因型活斑马鱼幼虫的策略导致了变聚合酶链反应 (PCR) 效率15。虽然最近公布的显微尾部活检技术提供了更有希望的结果16, 我们和其他小组无法重现的高效率和 DNA 恢复报告的作者。威尔金森等所描述的协议的一个重大倒退。16可能是尾巴组织的转移到 PCR 管。由于其显微尺寸, 很难确保鳍被正确收集和配发的吹打。为了解决这一障碍, 我们开发了一种改进的协议, 用于对大量活斑马鱼幼虫进行基因分型, 其效率为 100%9。使用 microscalpel, 鳍尾的尖端被移除并放置在一张滤纸上。该片含有组织的滤纸可以很容易地可视化, 并正确地放入 PCR 管。然后进行基因组 DNA 提取, 然后进行 PCR 扩增的感兴趣区域基因型的标本。该方法的优点有: PCR 效率高, 假阳性率低, 死亡率低。另外, 使用该协议可以对大量胚胎进行基因分型。本文所述的协议允许在 2-3 小时内用这种方法对数以百计的幼虫进行翅片修剪, 并在两天内正确识别 genotyped 鱼和尾再生。

<table>
	<tbody>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscalpel</td>
			<td>World Precision Instruments</td>
			<td>500249</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine/Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Tissue-culture plate</td>
			<td>Costar</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate</td>
			<td>Fisher</td>
			<td>14230232</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher</td>
			<td>S25548A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Chelex 100 sodium form</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td></td>
		</tr>
		<tr>
			<td>GelRed 1x</td>
			<td>Biotium</td>
			<td>41003</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 system</td>
			<td>Bio-Rad&#160;</td>
			<td>1704508</td>
			<td></td>
		</tr>
		<tr>
			<td>30 % Acrylamide/Bis Solution, 37.5: 1</td>
			<td>Bio-Rad&#160;</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Green Master Mix, 2X</td>
			<td>Promega</td>
			<td>M7123</td>
			<td></td>
		</tr>
		<tr>
			<td>paper wipes (Kimwipes&#174; disposable wipers)</td>
			<td>Sigma-Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>1kb-plus molecylar weight marker&#160;</td>
			<td>Thermo Scientific</td>
			<td>SM1343</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 system (high-throughput agarose gel system)</td>
			<td>Bio-Rad&#160;</td>
			<td>1704508</td>
			<td></td>
		</tr>
		<tr>
			<td>vertical gel electrophoresis system Mini-PROTEAN Tetra Cell&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>1658004</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping

斑马鱼 (斑马斑马) 拥有 orthologues 已知与人类疾病相关的84% 的基因。此外, 这些动物有短的世代时间, 容易处理, 显示高繁殖率, 低成本, 并且容易地接受基因操作通过微注射 DNA 在胚胎。基因编辑工具的最新进展使斑马鱼的突变和转基因得到精确的引入。在斑马鱼的疾病建模往往导致幼虫表型和早期死亡, 这可能是挑战, 以解释, 如果基因的未知。在需要样本池的实验中也需要这种早期的基因型鉴定, 例如基因表达或质谱研究。然而, 广泛的基因型筛选受传统方法的限制, 在大多数实验室仅对成年斑马鱼或死后幼虫进行。我们解决这个问题的方法是, 采用一种分离 PCR 现成的基因组 DNA 从活斑马鱼幼虫, 可以达到早在72小时后受精 (hpf)。这一时间和成本效益的技术, 改进了从以前公布的基因分型协议, 允许鉴定的基因, 从微观鳍活检。随着幼虫的发育, 鳍迅速再生。然后, 研究人员可以利用这种高通量 PCR 的基因分型方法, 选择并提高所期望的成年期。

protocoll 的效率是通过基因分型的异型十字架 (aldh7a1+/ot100, 这里显示为aldh7a1+/) (图 3A-C)9。aldh7a1ot100突变等位基因有一个5基对 (bp) 插入的第一个编码外显子的斑马鱼aldh7a1基因导致 frameshift 和丧失功能, 由于早期停止密码子9。在多重反应中?...

Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

斑马鱼在生物医学研究中被用作可靠的遗传模型有机体, 特别是随着基因编辑技术的出现。当幼虫表型预计, DNA 提取和基因的鉴定可能是挑战。在这里, 我们描述了一个有效的基因分型程序的斑马鱼幼虫, 通过尾部修剪, 早在72小时后受精。

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

high-throughput-dna-extraction-genotyping-3dpf-zebrafish-larvae-fin

Research

JoVE Journal

Genetics

13.9K 次观看.  Children's Hospital of Eastern Ontario Research Institute. 斑马鱼在生物医学研究中被用作可靠的遗传模型有机体, 特别是随着基因编辑技术的出现。当幼虫表型预计, DNA 提取和基因的鉴定可能是挑战。在这里, 我们描述了一个有效的基因分型程序的斑马鱼幼虫, 通过尾部修剪, 早在72小时后受精。

视频: High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...