这项工作的经费由圣地亚哥大学、国家科学基金会研究生研究金方案、NIH 赠款 R01-HG007836 以及韩国科学、信息和通信技术和未来规划部提供。
早期版本的几个数字首次发表在 "Hoople, 动力局等。凝胶序列: 用半透水水凝胶屏障同时进行低输入 DNA 和 RNA 文库制备的全基因组和转录测序。实验室在芯片 17, 2619-2630, 对: 10.1039/c7lc00430c (2017)。芯片上的实验室已经批准了这个出版物中的数字重用。

1. 化学溶液制备
注: 以下步骤用于在以后的步骤中准备需要的化学溶液。这些可以批量制作, 储存几个月。
<ol><li>开始时, 用 254 nm UV 交联烤箱中的15分钟 (15 mJ/cm2总曝光) 杀菌, 制备50毫升纯净的去离子水, 以中和任何污染的 DNA。加热到37摄氏度, 以便在以下步骤中使用。</li>
	<li>使10毫升的40% 总 (T) 和3.3% 交联剂 (C) (29:1) 聚丙烯酰胺前体溶液。重3.867 克丙烯酰胺单体和0.133 克的双丙烯酰胺单体。结合并使体积达到10毫升与温暖纯净水和漩涡, 直到溶解。储存在室温下保护免受光照。 注: 预混40% 吨, 3.3% C (29:1) 聚丙烯酰胺溶液可在商业上购买。</li>
	<li>使10mL 的 50% T, 5% C 凝胶溶液。重4.750 克丙烯酰胺单体和0.250 克的双丙烯酰胺单体。结合和带来的体积高达10毫升使用温暖纯净水和漩涡, 直到溶解。储存在室温下保护免受光照。</li>
	<li>使10毫升的 50% (瓦特/v) 蔗糖溶液。将5克蔗糖添加到一个已毕业的气缸中, 并将温纯净水添加至总容积10毫升。漩涡, 直到溶解和贮存在室温下。</li>
	<li>使10毫升 10% APS (瓦特/v)。将1克过硫酸铵 (APS) 添加到一个已毕业的气缸。加入冷 (~ 4° C) 纯净的水由总容量10毫升和漩涡直到溶化。立即冻结在整除数200µL。</li>
</ol>2. 胶体序列盒制作
注: 凝胶序列最初是用直立盒制作的 (请参阅材料表以了解更多信息);但是, 该协议可以适应任何标准的凝胶电泳盒。
<ol><li>通过添加试剂, 在三单独的塑料管中制备凝胶前体, 如下面的表1所示。不要添加 ap 或 Tetramethylethylenediamine (TEMED), 直到按以下步骤进行定向。涡流成分彻底混合。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="2">填料凝胶前驱体</td>
			<td colspan="2">高密度凝胶前驱体</td>
			<td colspan="2">低密度凝胶前驱体</td>
		</tr>
<tr>
<td>40%T, 3.3%C 丙烯酰胺 Bisacrylamide 溶液</td>
			<td>1.6 毫升</td>
			<td>50%T, 5%C 丙烯酰胺 Bisacrylamide 溶液</td>
			<td>2.4 毫升</td>
			<td>40%T, 3.3%C 丙烯酰胺 Bisacrylamide 溶液</td>
			<td>0.6 毫升</td>
		</tr>
<tr>
<td>去离子水</td>
			<td>10.2 毫升</td>
			<td>去离子水</td>
			<td>1.0 毫升</td>
			<td>去离子水</td>
			<td>4.8 毫升</td>
		</tr>
<tr>
<td>蔗糖溶液 (50% 瓦特/v)</td>
			<td>2.6 毫升</td>
			<td>蔗糖溶液 (50% 瓦特/v)</td>
			<td>0.6 毫升</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>10X 三硼酸-EDTA</td>
			<td>1.6 毫升</td>
			<td></td>
			<td></td>
			<td>10X 三硼酸-EDTA</td>
			<td>0.6 毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>过硫酸铵 (10% 瓦特/v)</td>
			<td>104.0 µL</td>
			<td>过硫酸铵 (10% 瓦特/v)</td>
			<td>50.0 µL</td>
			<td>过硫酸铵 (10% 瓦特/v)</td>
			<td>39.0 µL</td>
		</tr>
<tr>
<td>TEMED</td>
			<td>6.0 µL</td>
			<td>TEMED</td>
			<td>1.0 µL</td>
			<td>TEMED</td>
			<td>2.2 µL</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>总容积</td>
			<td>16.1 毫升</td>
			<td>总容积</td>
			<td>4.1 毫升</td>
			<td>总容积</td>
			<td>6.0 毫升</td>
		</tr>
</tbody></table>表1。凝胶合成试剂.聚丙烯酰胺凝胶前体试剂足以制造2盒磁带。
<ol start="2"><li>脱气凝胶单体解决方案通过插入一根针通过管帽, 并连接到一个房子的真空线针。将此组件浸入到高的超声波浴缸中, 等到气泡停止从液体中出现, 然后再移动到下一步 (~ 60 秒/管)。 注意: 只要气泡能从溶液中浮现出来, 超声波浴的真空和功率质量就不重要了。</li>
	<li>将 TEMED 和 APS 添加到填料凝胶前驱体和旋涡中。立即添加6毫升的填料凝胶前体的每一个凝胶盒通过吹打的解决方案到盒顶部。使用1毫升微添加前体六增量, 以避免溢出。此步骤的总时间应少于3分钟。</li>
	<li>通过将卡带直立放置在水平表上, 确保流体水平。用去离子, 脱气水填满卡带的其余部分。吸管的水, 再次使用1毫升的增量, 慢慢进入中心的卡带, 以尽量减少混合。允许聚合物固化至少一个小时, 直到一夜之间。</li>
	<li>在聚合物固化后, 将凝胶盒在水槽上反转, 以除去水覆盖物。压缩空气枪可以用来轻轻地干燥界面, 通过吹空气通过顶部打开的卡带从6英寸的距离。</li>
	<li>将 TEMED 和 APS 添加到高密度凝胶前驱体和涡流中。立即添加320µL 的高密度前驱物的卡带, 再次使用1毫升吸管。确保流体均匀涂层填充凝胶层通过摇摆磁带来回3倍左右。此步骤应少于三分钟。</li>
	<li>用去离子, 脱气水填满卡带的其余部分。吸管慢慢地用1毫升的吸管进入卡带的中心, 以尽量减少混合。允许聚合物固化至少十五分钟, 最好是一个小时。</li>
	<li>再次, 反转胶盒, 以消除水覆盖。压缩空气可以用来轻轻地烘干界面。将 TEMED 和 APS 添加到低密度凝胶前驱体和旋涡中。立即用低密度凝胶前驱体填充卡带 (~ 1.65 毫升) 的其余部分, 然后插入凝胶梳。</li>
	<li>在梳子的顶端, 吸管的过剩的储备前驱, 因为它将被吸收在聚合过程中。允许聚合物固化至少4小时, 最好是过夜。凝胶在三硼酸-乙二胺 (EDTA) 缓冲液中可贮存一周或更多次。</li>
</ol>3. 样品准备和反向转录
<ol><li>从感兴趣的细胞悬浮开始, 并在聚合酶链反应 (PCR) 层流罩中工作, 使用 hemocytometer 或自动细胞计数器计算细胞浓度。在磷酸盐缓冲生理盐水 (PBS) 中, 将细胞稀释成每µL 100 到1000个细胞的浓度。 注意: 此协议已在包括 PC3、HeLa 和小鼠肝细胞在内的一系列细胞中得到验证。</li>
	<li>使用在µL 试剂盒中提供的试剂 (参见材料表), 混合19的裂解缓冲液和1µL 的 RNase 抑制剂, 以制备反应缓冲器的10X 库存溶液。创建一个包含0.5 µL 反应缓冲器和2.75 µL 核酸酶自由水的溶解主混合。</li>
	<li>将细胞悬浮液移至5次, 以重新悬浮已沉淀的细胞, 然后将1µL 的样品注入到200µL 核酸酶自由条管中, 紫外线杀菌。根据样品的数量, 必要时重复。一定要包括一个负控制吹打1µL 核酸酶自由水, 而不是细胞的一个反应。接下来, 添加3.25 µL 的裂解主混合到每个样品和混合通过轻轻吹打上下5次。</li>
	<li>预热热循环仪 (带加热盖子) 到72摄氏度。添加1µL 的 RT 底漆和1µL 20 µM 随机水溶液与 WTA 适配器 (5′ AAGCAGTGGTAT-CAACGCAGAGTAC-NNNNNN 3′) 的每个样本。保留至少一管作为一个积极的控制 gDNA 图书馆的准备和添加2µL 的水, 而不是底漆。 注: 带有水溶液适配器的随机使用是可选的, 对排序结果的影响最小。</li>
	<li>在预热的热循环仪中孵育72摄氏度的样品, 3 分钟溶解细胞。从热循环仪中取出细胞, 在冰上放置2分钟。将正控制存储在4摄氏度, 直到步骤5。</li>
	<li>当细胞株溶藻时, 为所有含有以下试剂比率的 RNA 样本创建一个足够数量的反向转录主混合体: 2 µL 第一链缓冲器、0.5 µL 模板开关寡核苷酸 (草)、0.25 µL RNase 抑制剂和1µL逆转录酶 (100 U/µL)。</li>
	<li>预热热循环仪到42摄氏度。添加3.75 µL 反向转录主混合到其余的样本, 使总样本量为10µL. 混合吹打上下5次。</li>
	<li>立即将样品放在预热的热循环仪中进行反向转录。运行以下程序:42 °c 为90分钟, 70 °c 为10分钟, 4 °c 永远。这是一个安全的停车点。</li>
</ol>4. 凝胶分离和样品回收
<ol><li>使用 DNA 去除产品仔细清洁凝胶电泳室。将液体清洗剂的几个毫升应用于一次性的皮棉免费擦拭, 并在房间的所有表面擦拭, 然后用干净的0.5x 填充房间。为获得最佳效果, 请将整个设备放置在 254 nm UV 交联烤箱中, 并杀菌15分钟 (15 mJ/cm2)。</li>
	<li>将凝胶序列盒插入凝胶电泳室并将其锁定到位。慢慢取出凝胶梳, 直拉。缓慢移动以避免撕裂凝胶或撕裂任何武器。</li>
	<li>将样本从步骤3保留在冰上, 至少保留一个样本作为对 cDNA 文库生成的积极控制。将此控件存储在4摄氏度, 直到步骤6。将2µL 的6X 负载染料添加到剩余的样品中, 使总容积达到 ~ 12 µL. 通过吹打上下5次完全混合样品。</li>
	<li>结合1µL 的 DNA 阶梯与2µL 的6x 负载染料和7µL 的水。将这种混合物注入凝胶序列盒中的1车道作为电泳控制。将前一步的样品移到凝胶序列盒的不同车道上。小心地防止水井之间的污染, 把吸管充分插入每个井, 并删除它只使用垂直运动。</li>
	<li>使用标准的凝胶电泳电源, 在凝胶序列盒中应用 250 V 的电场30分钟, 将 gDNA 与 cDNA/RNA 混合体分开。一旦分离, 从凝胶电泳室中取出凝胶序列盒, 并用刮边工具撬开磁带的两半部分。</li>
	<li>用手术刀, 把凝胶切成两半, 就在高密度层下面。用戴手套的手捡起来, 丢弃含有填充胶的那一半。用刮刀或其他类似的工具, 轻轻地剥开卡带上剩下的凝胶。将凝胶的这一部分放入含有30毫升0.5x 的盘中, 3 µL 凝胶染色。</li>
	<li>盖上容器, 尽量减少漂白和浸泡凝胶, 同时轻轻摇动容器5分钟。将凝胶放在塑料包装上, 并使用凝胶文档系统进行 UV 图像 (有关进一步详细信息, 请参阅参考文献 13)。30秒的曝光通常会产生清晰的图像。验证是否已发生分离。</li>
	<li>将凝胶移至紫外线 transilluminator, 以促进核酸的可视化。佩戴合适的 UV 眼镜, 确认凝胶文件系统的结果。gDNA 应位于低密度凝胶的起始处和低密度高密度区域界面的 cDNA。</li>
	<li>使用手术刀, 切除含有 gDNA 和 cDNA 的凝胶区域。最好通过切割4毫米10毫米的凝胶矩形段来回收样品;然而, 确切的几何将取决于使用的凝胶电泳系统。记住还要切断带有负控制的车道。</li>
	<li>使用一对钝端镊子将凝胶的每个切除部分放入条带管中。小心不要施加太多的力量或凝胶将分裂成多个片断。如果发生这种情况, 只需拿起每一块, 并添加到管。</li>
	<li>用吸管的尖端 (200 µL 吸管提示) 在每管中研磨凝胶, 将吸管尖端以圆形的方式移动到管子的底部。将核酸酶免费水 (40 µL 进入 gDNA 样品和80µL 到 cDNA 样品中), 然后除去用于研磨凝胶的吸管尖端, 以减少样品损失。</li>
	<li>把带管的漩涡搅拌机内的37°c 孵化器和震动 8-12 小时。这使得核酸能从凝胶中扩散出来, 是这个多天协议的自然停止点。</li>
	<li>将样品注入8µm 网格过滤板, 在 2600 x g 处旋转板5分钟以应变出凝胶碎片。将网格过滤板从外壳板上提起, 将无凝胶水样注入新的200µL 条管。</li>
	<li>添加1µL 蛋白酶 (0.9 AU/毫升) 的每个样本包含 gDNA, 混合良好的吹打上下, 并孵化50°c 15 分钟后, 热失活在70°c 15 分钟。此步骤对耗尽核非常重要, 并使 gDNA 可用于后续反应步骤。</li>
	<li>使用18口径的针, 戳孔在所有样品管的上限。将样品放在 vacufuge 中以减少液体体积。gDNA 样品应减少到5µL 和 cDNA 样本减少到10µL。</li>
	<li>根据样品的 vacufuge 和数量, 总蒸发时间将在30和60分钟之间变化。如果样品体积低于目标体积, 只需添加核酸酶自由水增加样本量。</li>
</ol>5. gDNA 图书馆的准备工作
<ol><li>使用荧光计或类似技术, 从步骤4中量化每个 gDNA 样本中的 DNA 浓度, 以及步骤3中的正向控制。有关详细协议, 请参阅荧光计参考手册。14
</li>
	<li>将样品稀释到0.2 µL 的 DNA。根据所需的起始细胞类型和结果质量, 较低浓度仍可能产生可行的库。将需要进行一些实验;然而, 作者的成功与图书馆的低至 0.1 ng/µL。</li>
	<li>完成 gDNA 库准备工作, 按照库准备步骤7中的半反应卷协议。</li>
</ol>6. cDNA 文库准备
<ol><li>从步骤4开始, 10 µL cDNA 样本和阳性对照, 添加12.5 µL 2X qPCR 混合, 0.5 µL cDNA PCR 底漆, 2 µL 无核酸酶水。</li>
	<li>使用以下协议在实时 thermocycler 中执行 PCR: 热启动在95°c 为3分钟, 其次20-30 个周期98°c 为十年代, 65 °c 三十年代和72°c 为3分钟. 总样本量为25µL. 监测反应曲线, 停止放大前e 反应离开指数相位 (线性信号增加与周期数), 以避免 PCR 工件由于 overamplification。有关避免 overamplification 的更多内容, 请参见本文的讨论部分, 以及参考文献15。</li>
	<li>放大后, 使用固相可逆固定 (SPRI) 珠在步骤8中的协议后清洁产品。完成后, 继续执行下一步。</li>
	<li>使用荧光计或类似技术, 量化每个 cDNA 样本中的 DNA 浓度以及步骤4中的阳性对照。如果需要, 稀释样品, 以包含大约0.2 µL 的 DNA。稍低的浓度仍然产生可行的库。一些实验将是必要的, 然而作者有成功与图书馆低到 0.1 ng/µL。</li>
	<li>可选步骤: 对每 qPCR 产品的 1-2 µL 进行标准的聚丙烯酰胺凝胶电泳分离, 以验证库代反应工作。图 4中显示了成功结果的示例图像。有关如何进行聚丙烯酰胺凝胶电泳的详细协议, 请参阅参考文献16。</li>
	<li>完成 cDNA 库准备工作, 遵循库准备套件在步骤7中的半卷反应协议。</li>
</ol>7. 具有半容积反应的图书馆准备工作
<ol><li>图书馆准备工作遵循图书馆准备套件协议使用半容积反应。17协议本节中引用的所有试剂都来自库准备工具包 (请参阅材料表)。开始由紫外线杀菌足够数量的带管的数量的样品要处理。</li>
	<li>transposase 反应通过增加5µL transposase 缓冲到每条管将用于化验。然后添加2.5 µL 输入 DNA 0.2 ng/µL (0.5 吴总) 后跟2.5 µL transposase。混合吹打上下5次。</li>
	<li>孵育55°c 5 分钟, 然后保持在10摄氏度。样品达到10摄氏度后, 将其从 thermocycler 中取出, 并立即向每个样品添加2.5 µL transposase 停止缓冲器。把样品放在室温下5分钟。</li>
	<li>通过添加7.5 µL 库准备 pcr 组合, 2.5 µL 指数1底漆, 2.5 µL 指数2底漆, 制备 pcr 反应放大转置处理样品。这些底漆是专有的, 由图书馆准备工具的制造商提供。混合吹打上下5次。 注: 确保每个样品使用独特的底漆组合。有12不同的索引1引物和8不同的索引2底漆, 使成为可能唯一标签多达96个不同的样品。为每个样品选择一个独特的底漆组合。</li>
	<li>使用以下程序在 thermocycler 上执行 PCR。样品体积为25µL。 72°c 3 分钟 95°c 30 秒 12周期: 95°c 10 秒 72°c 30 秒 55°c 30 秒 72°c 5 分钟 保持在10°c</li>
	<li>按照步骤8中的协议, 使用 SPRI 珠子清理准备好的库。这些图书馆应该使用凝胶电泳或类似的化验来验证。有关如何验证库的详细信息, 请参阅库准备工具包手册。17
</li>
</ol>8. 固相可逆固定化珠库清洗
<ol><li>固体相可逆固定化 (SPRI) 珠应储存在1.5 毫升整除数, 并应带到室温, 每次使用。还建议为每个实验准备新鲜的80% 乙醇。以下步骤基于 "SPRI" 中的 "球珠协议" 手册。18
</li>
	<li>在每种 PCR 产品中添加1µl 的包裂解缓冲液。涡流 SPRI 珠均匀混合, 然后添加50µl 的 SPRI 珠每个样品。混合通过吹打样品上下10次, 然后在室温下孵化样品8分钟。</li>
	<li>简单地旋转样品, 从管子的侧面收集液体。将样品放在磁分离装置上5分钟, 直到液体完全清晰为止。</li>
	<li>当样品在磁性分离装置, 慢慢地去除上清和放弃-小心不要打扰在管子的珠子圆环。接下来添加200µL 80% 乙醇的每个样品, 而不干扰珠子。等待30秒钟, 然后仔细地吸出上清。重复此步骤 (乙醇洗涤) 一次。</li>
	<li>允许样品干燥三十年代-1 分钟。不要过度干燥的样品, 因为大碎片将成为永久性绑定到珠子。 注意: 建议简单的干燥时间, 以确保所有的乙醇的痕迹被删除。如果微量的乙醇留在后面, 它们可以略微抑制下游反应。</li>
	<li>从磁分离装置中取出样品, 并将15µL 的水添加到每个样品中, 以洗脱珠子上的 DNA。吸管向上和向下, 并确保珠子从管的两侧被删除。在室温下孵育2分钟。简单地旋转样品, 然后将它们放在磁分离装置上1分钟, 直到溶液看起来清晰为止。</li>
	<li>当样品在磁分离装置上时, 慢慢地将上清液吸干, 并将其转移到清洁管上。小心不要打扰管上的珠子环。用珠子把管子扔掉。</li>
</ol>

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtypes+and+diversity+revealed+by+single-nucleus+RNA+sequencing+of+the+human+brain.">Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain.</a> Science. 352 (6293), (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Qubit 3.0 Fluorometer Manual. MAN0010866. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+thousands+of+single-cell+genomes+with+combinatorial+indexing.">Sequencing thousands of single-cell genomes with combinatorial indexing.</a> Nat Methods. 14 (3), 302-308 (2017).</li><li>Illumina. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nextera &#174; XT DNA Library Preparation Kit. , (2017).</li><li>Takara Bio USA Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing User Manual. , (2016).</li><li>Kurimoto, K., Yabuta, Y., Ohinata, Y., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+single-cell+cDNA+amplification+to+provide+a+template+for+representative+high-density+oligonucleotide+microarray+analysis.">Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis.</a> Nat Protoc. 2 (3), 739-752 (2007).</li></ol>

KZ 是 Singlera 基因组公司的共同创始人和科学顾问。

有几个关键步骤相关的凝胶序列设备的制造以及协议本身。在制作过程中, 我们建议从规定的层厚度开始, 为凝胶的各个区域。我们花了大量的时间测试不同的制造选项, 此处描述的协议为材料和试剂表中列出的磁带制作最佳设备。如果研究人员使用另一种卡带系统, 他们可能会发现有必要调整在创建设备时使用的卷。制造中的主要挑战是, 如果高密度凝胶区域太大, 它可以从卡带的边?...

下一代测序技术对遗传学研究的实施方式产生了深远的影响。研究人员曾经专注于对整个物种的基因组进行排序, 现在可以在一个实验中序列化单个肿瘤的基因组, 甚至单个细胞。1转录还使在单元格中找到的 RNA 记录集 (称为 "数据收集") 的序列化具有成本效益。在过去的五年里, 从小的起始样本中放大和序列 DNA 或 RNA 的能力才得以实现。2,3,4不幸的是, 标准协议是不兼容的, 研究人员必须选择是否为给定的样本序列 DNA 或 RNA。当一个起始样本足够大时, 它可以分成两半。然而, 在较小的尺度上, 由于分裂样本造成的材料损失会影响到图书馆的质量, 而样品的汇集可以平均出细胞之间的有趣变化。5此外, 研究人员越来越热衷于检查不能分割的样本, 如单细胞或小的异质肿瘤活检。6
为了解决这一问题, 最近开发了三项协议, 用于从同一起始样本中序列化 DNA 和 RNA: 凝胶顺序7、G & t-序列8和 DR-序列9。本文给出了凝胶序列的详细协议, 可用于同时生成少量100细胞的 DNA 和 RNA 库, 其成本可以忽略不计。凝胶序列的新方面是将 DNA 和 RNA 完全用低成本水凝胶基质分离出来的能力。凝胶序列协议的核心创新是 DNA 与 RNA 的物理分离。这种分离是实现电泳使用聚丙烯酰胺膜的组合, 利用这些分子之间的大小差异。要将这些大小差异放在上下文中, 请考虑 dna 和 RNA 是如何成像的: 虽然 dna 存在于微米尺度上, 可以用传统显微镜观察, 但 RNA 存在于纳米尺度上, 必须使用诸如低温电子等复杂技术进行成像。显微镜.10
在这个协议中分离 DNA 和 RNA 的方法如图 1所示。左面板显示 DNA 和 RNA 游离漂浮在溶液附近的膜。当电场被应用时, 如右面板所示, DNA 和 RNA 经历了一种电泳力, 通过细胞膜诱导迁移。通过调整膜的性能, 我们创造了一种半渗透膜, 将 DNA 与 RNA 分离。DNA 分子被推到膜上, 但由于它们的体积大而被边缘纠缠。小 RNA 分子, 另一方面, 可以重新配置和编织他们的方式通过膜。这个过程, 被称为蠕动, 类似于蛇在草丛中穿行的方式。最终, 这些 RNA 分子被第二个高密度膜所阻挡, 对于更小的聚合物 (> 200 基对) 来说, 这是非常困难的。一旦物理分离, DNA 和 RNA 可以被恢复和处理, 以产生关于基因组和转录的信息。虽然我们可以分离 DNA 和 rna, 我们发现更好的结果, 如果 RNA 反转转录为 cDNA 在分离之前。cDNA/rna 杂交种比 rna 更稳定, 还能通过低密度膜。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57315/57315fig1.jpg"> 图 1.凝胶的操作原理.用于物理分离 DNA 和 RNA 的基本原则。在应用电场中, 小 RNA 分子通过低密度膜迁移, 但大量的 DNA 分子被困在表面。这个数字是从参考文献7中转载的, 得到了皇家化学学会的许可。<a href="//cloudflare.jove.com/files/ftp_upload/57315/57315fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
本文详细介绍了凝胶序列器件的制备和生成配对 DNA 和 RNA 库的生物协议。这两者的概览都显示在图 2中。该装置是通过分层三种不同密度的聚丙烯酰胺凝胶在一个类似于创建标准的堆叠凝胶的过程中制造的。11生物协议从 PBS 中悬浮的 100-1000 细胞开始。细胞被裂解, RNA 被转化为 cdna, 然后用该装置将基因组 DNA 与 cdna/RNA 杂交体分离。在分离和恢复后, 基因组和 transcriptomic 库是使用一个密切遵循标准全基因组库准备套件协议的过程编写的。关于凝胶序列的发展和验证的进一步细节可以在实验室中读到一个芯片出版物"凝胶序列: 全基因组和转录测序通过同时低输入 DNA 和 RNA 库准备使用半透水水凝胶屏障."7 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57315/57315fig2.jpg"> 图 2.凝胶序列协议.概述了制造凝胶序列装置的步骤和生成配对 DNA 和 RNA 库的协议。这个数字的部分从参考文献7被复制了从皇家化学学会的允许。<a href="//cloudflare.jove.com/files/ftp_upload/57315/57315fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了从单细胞中生成 DNA 和 rna 库, 研究人员应该考虑使用 g & t 序列或 DR-下克 & t 序列, 如凝胶序列, 依赖于从基因组 DNA 中提取 rna 的物理分离。这种方法依靠信使 RNA 的 (mRNA) 3′ polyadenylated 尾巴作为下拉目标。该 mRNA 使用生物素化寡核苷酸底漆在磁珠上捕获。一旦基因被捕获, 珠子就会被放置在一个磁铁上, 含有基因组 DNA 的上清液可以被移除并转移到另一管。在这种物理分离完成后, 可以从 mRNA 和 DNA 中生成独立的库。8如果感兴趣的 RNA 是 polyadenylated, 则此方法很有效, 但是它不能用于研究非 polyadenylated 转录, 如核糖体 rna、tRNA 或原核生物中的 rna。
DR 序列依赖于一个预放大的步骤, 从 RNA 中提取的 DNA 和 cDNA 在同一管中被放大。然后将该样本分成两个, 并并行处理以准备 DNA 和 RNA 序列库。为了区分基因组 DNA 和 RNA 衍生的 cDNA, DR 序列采用了计算方法。只有外显子存在的序列在基因组 DNA 数据中被计算抑制, 因为它们可能来源于 DNA 或 RNA。9这种方法的优点是, DNA 和 cDNA/RNA 不需要物理分离, 如在凝胶序列和 G & T。然而, 缺点是, 转录博士需要先验知识的基因组和 (即, 外显子与内含子), 并可能不理想的应用, 如核测序, 其中许多成绩单尚未完全拼接并仍包含内含子。12
凝胶序列的新的方面是能够分离 DNA 和 RNA 在数以百计的细胞完全基于大小。此方法不需要先验知识的基因组或转录, 是强大的反对不完全剪接, 并不仅限于多 adenylated 成绩单。对于研究人员可以从至少100个细胞开始的应用, 凝胶序列提供了一个简单的方法使用廉价和广泛可用的材料。

<table border="0" cellpadding="0" cellspacing="0" style="width:905px;" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Reagents</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acrylamide Monomer</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A8887-100G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ammonium Persulfate</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A3678-25G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Ampure XP Beads</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td style="width:369px;">Referred to in the text as solid phase reversible immobilization (SPRI) beads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">DNA Gel Loading Dye (6x)</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">R0611</td>
			<td style="width:369px;">Referred to in the text as 6X loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ethyl alcohol</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">E7023-500ML</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">KAPA SYBR FAST One-Step qRT-PCR Kits</td>
			<td style="width:147px;">Kapa BioSystems</td>
			<td style="width:119px;">7959613001</td>
			<td style="width:369px;">Referred to in the text as 2X qPCR mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">N,N&#8242;-Methylenebis(acrylamide)&#160;</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">146072-100G</td>
			<td style="width:369px;">Also known as bis-acrylamide</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:271px;">NexteraXT DNA Library Preparation Kit (referred to in the text as library preparation kit)</td>
			<td style="width:147px;">Illumina</td>
			<td style="width:119px;">FC-131-1024</td>
			<td style="width:369px;">Includes: TD (referred to in the text as transposase buffer), ATM (referred to in the text as transposase), NT (referred to in the text as transposase stop buffer), and NPM (Referred to in the text as library prep PCR mix)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Nuclease Free Water</td>
			<td style="width:147px;">Millipore</td>
			<td style="width:119px;">3098</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Protease</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">19155</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:271px;">SMART-Seq v4 Kit (referred to in the text as whole-transcript amplification (WTA) kit)</td>
			<td style="width:147px;">Takara/Clontech</td>
			<td style="width:119px;">634888</td>
			<td style="width:369px;">Includes: Lysis buffer, RNase inhibitor, 3&#8217; SMART-Seq CDS Primer II A (referred to in the text as RT primer), 5X Ultra Low First Strand Buffer (referred to in the text as first strand buffer), SMART-Seq v4 Oligonucleotide (referred to in the text as template switch oligonucleotide (TSO)), SMART-Scribe Reverse Transcriptase (referred to in the text as reverse transcriptase), and PCR Primer II A (referred to in the text as cDNA PCR primer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Random hexamer with WTA adapter&#160;</td>
			<td style="width:147px;">IDT</td>
			<td style="width:119px;">n/a</td>
			<td>5&#8242;-AAGCAGTGGTATCAACGCAGAGTAC-NNNNNN-3&#8242;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sucrose</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">S0389-500G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">TEMED</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">T9281-25ML</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">DNA AWAY Surface Decontaminant</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">7010PK&#160;&#160;</td>
			<td style="width:369px;">Referred to in the text as DNA removal product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Tris-Borate-EDTA buffer (10X concentration)</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">T4415-1L</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">SYBR Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO)</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11494</td>
			<td style="width:369px;">Referred to in the text as gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;"></td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">BD Precisionglide&#160;syringe needles, gauge 18</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">Z192554</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Branson CPX series ultrasonic bath</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">Z769363</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Empty Gel Cassettes, mini, 1.0 mm</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">NC2010</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Mesh Filter Plate - Corning&#160;HTS Transwell 96 well permeable supports - 8.0 &#181;m pore size</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">CLS3374</td>
			<td style="width:369px;">Referred to in the text as 8 um mesh filter plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PowerPac HC Power Supply</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1645052</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Qubit Fluorometer</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">Q33216</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vacufuge Concentrator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">22822993</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">XCell SureLock Mini-Cell system</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EI0001</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Bio-Rad CFX96 Touch Real-Time PCR Detection System</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1855195&#160;</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Amersham UVC 500 Ultraviolet Crosslinker</td>
			<td style="width:147px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">UVC500-115V</td>
			<td style="width:369px;">Discontinued, any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Gel Doc XR+ Gel Documentation System</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1708195</td>
			<td style="width:369px;">Referred to in the text as gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Dark Reader Transilluminator</td>
			<td style="width:147px;">Clare Chemical Research</td>
			<td style="width:119px;">DR89</td>
			<td style="width:369px;">Referred to in the text as UV transilluminator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">&#160;Ultrasonic&#160;Bath</td>
			<td style="width:147px;">Bransonic</td>
			<td style="width:119px;">1207K35</td>
			<td style="width:369px;">Any equivalent ultrasonic bath is acceptable.</td>
		</tr>
	</tbody>
</table>

gel-seq: a method for simultaneous sequencing library preparation of dna and rna using hydrogel matrices

在过去的五年里, 从小的起始样本中放大和序列 DNA 或 RNA 的能力才得以实现。不幸的是, 生成基因组或 transcriptomic 库的标准协议是不相容的, 研究人员必须选择是否为特定的样本序列 DNA 或 RNA。凝胶序列解决了这个问题, 使研究人员可以同时准备的 DNA 和 RNA 的图书馆从 100-1000 细胞使用一个简单的水凝胶装置。本文给出了该装置的制作方法, 以及生成配对库的生物协议。我们设计的凝胶序列, 使它可以很容易地执行其他研究员;许多遗传学实验室已经有必要的设备, 以重现凝胶序列的设备制造。我们的协议使用常用的工具包进行全文本放大 (transcriptomic) 和库准备, 这对于已经精通生成基因组和库的研究人员也很有可能熟悉。我们的方法使研究人员能够在不分裂和微不足道的附加成本的情况下, 在单个样品上携带 DNA 和 RNA 序列的能量。

通过荧光凝胶成像, 可以直观地分析凝胶序列中 gDNA 和 cDNA/RNA 混合体的物理分离;有代表性的结果显示在图 3中。面板 A 显示制备的凝胶序列装置;添加了假颜色来区分不同的凝胶区域。面板 B 显示了用于验证的四种不同分色的特写。第三条车道, 一个负控制, 代表背景, 并表明没有 autoflourescence 的凝胶在界面上。我们用 DNA 梯子装载了第一和第二条车道?...

Watch this Scientific Journal Video about Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices at JoVE.com

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

凝胶序列使研究人员能够同时为 DNA 和 RNA 的存储提供从 100-1000 细胞开始使用简单的水凝胶装置的可忽略的额外成本。本文给出了该装置的制作方法, 以及生成配对库的生物协议。

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

gel-seq-method-for-simultaneous-sequencing-library-preparation-dna

Research

JoVE Journal

Bioengineering

8.9K 次观看.  University of San Diego. 凝胶序列使研究人员能够同时为 DNA 和 RNA 的存储提供从 100-1000 细胞开始使用简单的水凝胶装置的可忽略的额外成本。本文给出了该装置的制作方法, 以及生成配对库的生物协议。

视频: Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

用于全基因组 DNA 分析的超长读数测序

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...