这项工作得到了 NIH 资助 P50HG004952 和 R01GM109099 的支持。

1. 捕获寡核苷酸设计
<ol><li>
设计在目标区域/s 的杂交捕获期间使用的寡核苷酸小组。
	<ol>
<li>目的设计每个目标区域的4–8寡核苷酸, 但最低限度, 设计至少一个针对目标区域的每一端的寡核苷酸。</li>
		<li>如果目标序列是长的 (> 500 基对), 设计出尽可能分散的寡核苷酸, 以确保在整个目标区域有效地丰富染色质。 注意: 我们观察到了4–8寡核苷酸的最佳捕获。虽然这个过程可以很好地与更多的寡核苷酸, 观察在大量寡核苷酸被使用时的富集率下降是罕见的。</li>
	</ol>
</li>
	<li>设计类似熔融温度的寡核苷酸, 确保它们不形成强发夹, 也不具有较强的交互作用 (考虑到杂交将在42ᵒC 进行)。 注: 如果选择了立足点洗脱 (4.12.3), 则必须将额外的外部8基序列添加到寡核苷酸中, 并且在洗脱17时需要互补的 "释放" 寡核苷酸。一些商业软件工具可以用来选择和分析寡核苷酸。25的市场汇率已经显示出良好的效果, 但根据基因组的大小和序列, 捕获寡核苷酸可以包含在20–80基地之间的某处。通常, 捕获寡核苷酸的熔化温度接近62ᵒC, 但这可能会改变, 取决于它们的长度。一致的熔化温度和/或寡核苷酸长度在整个捕获组合可以帮助避免不必要的偏见, 在浓缩。</li>
	<li>设计捕获寡核苷酸与生物素基团 (最好是分离的间隔从捕获序列) 在 3 ' 年底。</li>
</ol>2. 细胞培养
<ol><li>
生长人类永生细胞 (如此处显示的 GM12878) 或其他细胞系在 RPMI 1640 培养基中补充1% 青霉素-链霉素, 2 毫米 l-谷氨酰胺和5% 胎牛血清 (血清), 37 ᵒC 和 5% CO2。
	<ol>
<li>种子初始冷冻整除数 (2–5 x 10 5 细胞) 在 T-25 瓶中, 6 毫升 RPMI 1640 介质在2.1 中制备.</li>
		<li>监测细胞密度和活力。<ol>
<li>取一个具有代表性的细胞整除, 用合适的染料如台盼蓝染色。</li>
			<li>使用 hemocytometer 或自动单元格计数器测量细胞密度。</li>
		</ol>
</li>
		<li>在细胞达到 1 x 106细胞/mL 密度之前, 将细胞向下旋转 100 x g 以达到5分钟, 并将细胞转移到一个 T-75 烧瓶中, 25 毫升的 RPMI 1640 介质在2.1 中制备。将细胞生长直到细胞密度接近 1 x 106细胞/mL。</li>
		<li>将细胞转移到一个 T-150 烧瓶, 在38毫升的 RPMI 1640 培养基 (2.1), 并增加两天的细胞。 注: 永生细胞在悬浮液中生长。生长永生细胞的一般准则建议在 0.2–1 x 106细胞/mL 之间保持细胞密度。由于这项技术所需的大量材料, 本议定书的目的是将细胞生长到细胞密度之外。细胞生长协议可以由读者修改和修改, 具体取决于要使用的单元类型。</li>
		<li>以 100 x g 为5分钟旋转单元格, 并用重悬10毫升 RPMI 1640 介质 (2.1) 中的细胞, 然后将悬浮液倒入 850厘米2 滚轮瓶中, 其中包含500毫升的介质。孵化的细胞在相同的条件下, 但在这一点上开始使用恒定旋转 (~ 0.5 rpm)。</li>
		<li>监视细胞生长并在需要时改变介质 (通常每3–4天)。让细胞生长到密度尽可能接近 2 x 106单元格/mL (1 x 109单元格)。</li>
		<li>一旦达到所需的细胞计数, 通过将细胞向下旋转 100 x g 以5分钟并用重悬细胞, 并在36毫升的 RPMI 1640 培养基 (2.1) 中对细胞进行采集。将细胞悬浮液转移到50毫升锥形管上。用小整除数进行细胞计数和 DNA 提取。</li>
	</ol>
</li>
	<li>
交联通过加入1毫升37% 甲醛达到最后浓度的1%。 注意: 甲醛是一种致癌物, 应在通风罩中处理。<ol>
<li>在室温下 (RT), 用旋转 (~ 30–60 rpm) 孵化细胞10分钟。</li>
		<li>通过加入2毫升的2.5 米甘氨酸溶液来淬火交联反应, 最终浓度为125毫米。</li>
		<li>用旋转 (~ 30–60 rpm) 在 RT 上孵化细胞5分钟. 通过离心 100 x g 为5分钟, 用冰冷 1x PBS 清洗细胞两次。</li>
	</ol>
</li>
	<li>继续下一步或并用重悬颗粒在5毫升细胞裂解缓冲 (5 毫米 Hepes, 85 毫米氯化钾, 0.5% Igepal, 蛋白酶抑制剂 (PI)) 和抑制冻结的悬浮液滴下降的液氮。将样品存放在-80 ᵒC。 注: 准备 > 25 毫升的细胞裂解缓冲液没有 Igepal 和 PI, 并添加这些试剂新鲜时, 准备使用。一旦添加了 Igepal 和 PI, 避免了细胞溶解缓冲液的长期贮存。在4ᵒC 储存细胞裂解缓冲液长达3月。Igepal 是一种非离子的无变性洗涤剂。NP-40 可以作为 Igepal 的替代品, 但我们没有在这方面使用它, 因为当样品最终通过质谱分析来表征 DNA 结合蛋白时, 我们并没有用到 NP-40。</li>
</ol>3. 裂解和剪切
<ol><li>制备 > 15 毫升核裂解缓冲液 (10 毫米 EDTA, 50 毫米三 pH 8.0, 1% SDS, PI)。 注: 添加 PI 新鲜, 只是要使用的金额。一旦加入 PI, 避免了细胞核溶解缓冲的长期储存。核裂解缓冲液可储存在 RT 上长达1月。</li>
	<li>并用重悬20毫升细胞裂解缓冲液中的颗粒 (含 Igepal 和 PI)。让小球融化在冰上, 混合好一次解冻。让样品坐在冰上再加10分钟。离心细胞在 400 x g 在4ᵒC 5 分钟。</li>
	<li>放弃上清和并用重悬6毫升核裂解缓冲液中的细胞 (如果细胞不形成悬浮体, 体积可以增加)。</li>
	<li>
在准备超声波时, 将样品均匀地分在 ~ 6–8离心管 (每管600到1000µL)。
	<ol>
<li>将样品放在冰上或冷的架子上, 用 microtip 的 sonicator 油脂实验样品。使用65% 振幅与 5 x 二十年代恒定的爆发, 允许暂停降温至少四十年代之间的脉冲。 注: 如果体积增加 > 每样品10毫升, 可改用玻璃冷却电池。这时需要越来越多的爆裂。Sonicators 差别很大。条件可能必须由读者优化。其他超声波的替代品也可以使用。</li>
	</ol>
</li>
	<li>离心细胞在 1.2万 x g 10 分钟在4ᵒC, 转移上清到新的管, 并丢弃小球。</li>
	<li>使用荧光法估计总回收率。</li>
	<li>继续执行下一步或快照冻结, 并将该示例存储在-80 ᵒC。</li>
	<li>可选: 采取整除, 逆转 crosslinks 通过孵化它隔夜在67ᵒC 在0.3 米氯化钠。清除 DNA 并在 Bioanalyzer 中运行以确定片段长度。大部分的碎片应该在500和 1000 bp 之间。 注意: 此时停止并将样品存储在-80 ᵒC 是可以的。</li>
</ol>4. 杂交捕获
<ol><li>准备500毫升2x 杂交 (Hyb) 缓冲 (200 毫米 MES, 2M NaCl, 40 毫米 EDTA, 0.02% Tween-20) 和500毫升的洗涤缓冲器 (200 毫米氯化钠, 0.2% SDS, 50 毫米三 pH 值 8)。存储 Hyb 和洗涤缓冲器在4ᵒC 和室温, 分别为3月。使用 2x Hyb 缓冲区的一半来准备1x 杂交缓冲。还存储在4ᵒC 3 月。 注: 在上一步中, 杂交捕获的总体积将是结果样品的体积的两倍 (如前所述, 样品体积约为6毫升, 因此总杂交量为12毫升)。</li>
	<li>预留珠等于总杂交捕获量的 5% (在本例中为600µL)。在下面的步骤中, 需要一个合适的和合适的磁铁来处理珠子。</li>
	<li>用两次珠量 (1200 µL), 用 1x Hyb 缓冲器清洗珠子三次。并用重悬1200µL 2x Hyb 缓冲器中的珠子。将管放在磁铁上, 直到解决方案清除并卸下缓冲区。</li>
	<li>在1.5 毫升离心管中进行预清反应, 每管最大700µL 样品。添加足够的珠子 (5%) 和 2x Hyb 缓冲器稀释 Hyb 缓冲到 1x (10 管600µL 的样品, 120 µL 洗涤珠悬浮在 2x Hyb 缓冲器, 480 µL 的 2x Hyb 缓冲区)。孵化他们为10分钟在31ᵒC 与末端结束自转 (~ 30–60 rpm)。</li>
	<li>把管子放在磁铁上, 直到珠子固定并将样品转移到新的管子上。扔掉珠子。 注: 从这一点上, 建议低结合管。</li>
	<li>并用重悬捕获寡核苷酸到一个工作解决方案的 10 pmol/µL, 并添加4µL 这个解决方案, 每管。 注意: 不同区域可以相互控制, 但建议将捕获寡核苷酸包含在其序列上, 以作为一个真正的负控制。</li>
	<li>孵化管40分钟, 在42ᵒC 与端结束旋转 (~ 30–60 rpm)。</li>
	<li>在孵化期间, 预留所需的珠子 (0.3 µL/pmol 捕获寡核苷酸)。按照步骤4.3 中描述的三次清洗珠子, 但并用重悬 1 x Hyb 缓冲区中的单元格。</li>
	<li>将洗涤过的珠子添加到样品中, 并在 RT 上孵化出30分钟的样品以结束结束旋转 (~ 30–60 rpm)。把管子放在磁铁上, 一旦珠子固定了, 就把上面的清掉。</li>
	<li>
洗珠子
	<ol>
<li>添加1.2 毫升的洗涤缓冲和孵化的样本5分钟与结束结束旋转 (〜 30–60 rpm) 在 RT. 把管子放在磁铁上, 等几分钟, 直到溶液清除。删除缓冲区并重复此步骤。</li>
		<li>添加1.2 毫升的洗涤缓冲和孵化它为1小时与结束结束旋转 (~ 30-60 rpm) 在 RT. 把管子放在磁铁上, 等几分钟, 直到溶液清除。卸下缓冲区, 然后重复此步骤, 将潜伏期缩短到第二次30分钟。</li>
		<li>增加200µL 的洗涤缓冲和孵化它为15分钟以结束结束自转 (~ 30–60 rpm) 在31ᵒC。把管子放在磁铁上, 等几分钟, 直到溶液清除。删除缓冲区。</li>
		<li>添加200µL 的 PBS 和孵化它为5分钟与结束结束旋转 (~ 30–60 rpm) 在 RT. 把管子放在磁铁上, 等几分钟, 直到溶液清除。删除缓冲区并重复此步骤。</li>
	</ol>
</li>
	<li>
简单洗脱
	<ol>
<li>添加40µL 的分子级水的珠子和孵化它在94ᵒC 5 分钟。</li>
		<li>把管子放在磁铁上, 等几分钟, 直到溶液清除。</li>
		<li>把上清液转移到新的管子里。扔掉珠子。</li>
		<li>进行蛋白质组分析或将样品存储在-80 ᵒC。 注: 这种类型的洗脱工作很好, 为以后的 qPCR 分析和一些蛋白质组学测量, 但在其他情况下, 两种替代方法所描述的产量 "清洁" eluates。</li>
	</ol>
</li>
	<li>
DNase 洗脱。
	<ol>
<li>添加40µL 1x DNase 缓冲器和 2 U 的 DNase 每管。</li>
		<li>在37ᵒC 孵化试管15分钟。</li>
		<li>把管子放在磁铁上, 等几分钟, 直到溶液清除。</li>
		<li>从珠子中取出洗脱样品, 将样品放在新的低结合管中。</li>
		<li>将试管孵化94ᵒC 7 分钟, 使酶失效, 并进行蛋白质组分析。 注: 如果观察到对蛋白质组分析的干扰, 可以使用较低的 DNase 浓度。如果使用这种洗脱方法, DNase 处理完全破坏样品中的 DNA 材料, 就不可能计算 qPCR 的捕获充实。</li>
		<li>进行蛋白质组分析或将样品存储在-80 ᵒC。</li>
	</ol>
</li>
	<li>
立足点洗脱17。 注: 此方法不推荐用于较长的寡核苷酸 (> 40 碱基)。这种方法要求捕获寡核苷酸包含额外的8碱基, 不与基因组序列和互补释放寡核苷酸重叠, 以取代捕获寡核苷酸的目标。<ol>
<li>稀释 2 nanomoles 释放寡核苷酸在40µL 的 SSC 缓冲。</li>
		<li>在 RT 中孵化20分钟。</li>
		<li>把管子放在磁铁上, 等几分钟, 直到溶液清除。</li>
		<li>从珠子上取出溶液。</li>
		<li>进行蛋白质组分析或将样品存储在-80 ᵒC。</li>
	</ol>
</li>
</ol>5. qPCR 与引物对目标捕获区的俘获率的评价
<ol><li>使用适当的软件或在线工具设计目标区域的引物和探头。 注意: 在本实验中, DUSP3和DUSP5的启动子区域是目标对象。所用的引物和探头如下。DUSP3: 底漆 1-GTG TTT AGG TTC CCT, 底漆 2-CGG, TGC 铁通 CG, 家族标记探针-ATG CCC GAA。DUSP5: 底漆 1-热重 GAA AGC CCT GTG TC, 底漆 2-海湾合作委员会的 AAC CCC AAA AG, 家族标记探针 TGC 标签 agc agc, ATG AGG TT。</li>
	<li>取一个整除的洗脱液 (不超过10µL), 并稀释1:10 与分子级H2 O。</li>
	<li>提取步骤2.1.7 中的一个整除数的 dna, 并使用该 dna 样本通过在分子级 H 2 O 中做五系列1:10 稀释来制作标准曲线.</li>
	<li>准备一个 qPCR 主混合 (各种商业替代品), 添加底漆和探针, 并分配适当的数量 (15 µL, 如果运行一个20µL 反应) 在每个井。</li>
	<li>添加5µL 的两个: a) 稀释样品, b) 标准曲线或 c) 分子级 H2O (作为没有模板控制) 每个井。</li>
	<li>密封板和离心机在 100 x g 1 分钟。</li>
	<li>在具有以下参数的 qPCR 系统中运行: 5 分钟在95ᵒC 其次40周期95ᵒC 二十年代, 59 ᵒC 二十年代和72ᵒC 为二十五年代。</li>
	<li>使用标准曲线的驴产量和炒捕获, 以评估捕获特异性。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChAP-MS:+A+Method+for+Identification+of+Proteins+and+Histone+Posttranslational+Modifications+at+a+Single+Genomic+Locus.">ChAP-MS: A Method for Identification of Proteins and Histone Posttranslational Modifications at a Single Genomic Locus.</a> Cell Rep. 2 (1), 198-205 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+immunoprecipitation+(+eChIP+)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated immunoprecipitation ( eChIP ) using CRISPR.</a> Biochem. Bioph. Res. Co. 439 (1), 132-136 (2013).</li><li>Liu, X., 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=In+Situ+Capture+of+Chromatin+Interactions+by+Biotinylated+dCas9.">In Situ Capture of Chromatin Interactions by Biotinylated dCas9.</a> Cell. 170 (5), 1028-1043 (2017).</li><li>Myers, S. A., Wright, J., Zhang, F., Carr, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-APEX-mediated+proximity+labeling+enables+discovery+of+proteins+associated+with+a+predefined+genomic+locus+in+living+cells.">CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells.</a> bioRxiv. , (2017).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Proteins+Associated+with+Specific+Genomic+Loci.">Purification of Proteins Associated with Specific Genomic Loci.</a> Cell. 136 (1), 175-186 (2009).</li><li>Buxton, K. E., 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=Elucidating+Protein-DNA+Interactions+in+Human+Alphoid+Chromatin+via+Hybridization+Capture+and+Mass+Spectrometry.">Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.</a> J. Proteome Res. 16 (9), 3433-3442 (2017).</li><li>Kennedy-Darling, J., Holden, M. T., Shortreed, M. R., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+programmable+release+of+captured+DNA.">Multiplexed programmable release of captured DNA.</a> Chembiochem. 15 (16), 2353-2356 (2014).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+specific+genomic+regions+and+identification+of+associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Chromatin Protoc. Third Ed. , 43-52 (2015).</li><li>Lambert, J. -. 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没有任何利益冲突可供披露。

这里描述的 HyCCAPP 方法有许多独特的特点, 使它成为一个强有力的方法来揭示 DNA-相互作用, 否则将仍然难以捉摸。这个过程的性质使 HyCCAPP 在基因组的不同有机体和区域工作的灵活性。然而, 这是一个方法, 有几个限制要考虑。
HyCCAPP 是一种避免任何单元格修改的方法, 以便它可以在主细胞、细胞培养系统甚至组织样本中应用。然而, 由于这一原因, 它需要将样品交联, 从而降低蛋白质组分析中的灵敏度, 因此需要大量的输入量。有新的方法, 依靠 CRISPR 和 biotinylation 的周围的蛋白质, 可以功能不交联反应12,13,14,18。如果质粒插入不表示约束, 但只能在细胞培养系统中使用, 这些方法可能非常强大。其他方法非常适合于根据染色质状态或特定转录因子的存在来识别一般蛋白质协会, 但并不意味着研究单个的点9,10,19.
HyCCAPP 方法提出了一种具有广泛适用性的替代方法, 但在不同的细胞系统或生物体中应用可能需要优化。该方法最关键的优化步骤包括捕获寡核苷酸的设计和选择。一系列小规模的测试应该表明, 如果设计的寡核苷酸是适当的, 如果该区域的序列被有效捕获。在设计和测试捕获寡核苷酸时, 应考虑几个因素, 如寡核苷酸长度、目标区域的特异度以及要使用的寡核苷酸之间的相互作用。所有这些交互都可以通过使用特定于目标区域的 qPCR 检测来容易地进行测试。如果需要, 可以使用类似于芯片序列的工作流对生成的 DNA 片段进行排序, 并确保在整个基因组5中浓缩的特异性是令人满意的。
最后, 交联条件在 HyCCAPP 过程中也非常重要, 我们观察到不同系统之间的显著差异, 通过1% 甲醛的交联来获得人体细胞的最佳结果, 而在酵母中,交联与3% 甲醛产生了更重现的结果。这是可能的, 更激烈的交联与3% 甲醛在人类细胞与一个更复杂的染色质结构不提供足够的可获得性杂交捕获寡核苷酸。还可以想象, 酵母细胞壁的存在阻碍了甲醛进入细胞, 增加了在染色质中成功交联水平所需的甲醛量。
执行芯片和其他捕获方法以交联的人类染色质为目标, 我们观察到, 只有约1% 的目标 DNA 实际上可以被捕获。对于大多数基于 DNA 的方法和分析, 这不是一个限制因素。然而, 不像芯片, 最终读数是基于 amplifiable DNA, HyCCAPP 依赖于蛋白质含量的最终读数。由于这种内在的局限性, 需要大量的输入材料 (这里所提供的实验中的培养细胞): 在探索这种方法之前, 应仔细考虑这一限制, 特别是适用于单拷贝区域时。并非所有系统都能够生成所需的单元格数, 或者增加所需的单元格数的成本可能会令人望而却步。HyCCAPP 所需的输入金额 (~ 109单元格) 可与依赖交联材料的其他方法相媲美, 其输入量介于 108和 1011单元9、11、15之间。今后对 HyCCAPP 技术的修改将探讨人为地增加目标区域的拷贝数的方法, 以使此方法更广泛地适用。同时, 我们将努力继续提高整个过程的效率, 随着质谱的不断进步, 应该减少所需的输入量, 并使这一技术在更多的系统中可行。
使用 CRISPR 的基于 Biotinylation 的方法, 如 enCHiP18和其他13,14, 已证明是非常有效的, 消除了交联材料的需求, 增加产量, 并显著减少输入要求。这些方法中细胞的精细处理但是, 不允许这些技术应用于组织样本, 这是我们已经开始与 HyCCAPP 的方向, 这正在产生有希望的结果。

在过去的十年中, 测序技术有了显著的改进, 允许对大量样本中的各种基因组进行研究, 并具有惊人的分辨率。DNA 元素百科全书 (编码) 联合体是国家卫生研究院国家人类基因组研究所牵头的大规模多机构努力, 它提供了关于个人转录因子如何和其他调控蛋白结合在一起, 并与基因组相互作用。最初的努力的特点是特定的 dna 蛋白相互作用, 由染色质免疫沉淀 (芯片) 评估超过100已知的 dna 结合蛋白 1.替代方法, 如 DNase 足迹2和甲醛辅助隔离的管理元素 (自由放任)3也被用来定位特定区域的基因组相互作用的蛋白质, 但明显的局限性,这些实验方法不能识别相互作用的蛋白质。尽管过去几年进行了广泛的努力, 但没有任何技术能有效地使染色质中蛋白质-DNA 相互作用的综合特征, 以及染色质相关蛋白的鉴定和定量化。
为了解决这一需要, 我们开发了一种新的方法, 我们称之为杂交捕获的染色质相关蛋白的蛋白质组学 (HyCCAPP)。最初开发的酵母4,5,6, 该方法分离的交联染色质区域的利益 (与绑定蛋白) 使用序列特定的杂交捕获。在分离蛋白质-DNA 复合物后, 可以用质谱等方法来表征与兴趣序列有关的蛋白质集。因此, HyCCAPP 可以被认为是一种无偏见的方法来揭示新的 DNA-蛋白质相互作用, 在这个意义上, 它不依赖于抗体, 它是完全不可知的蛋白质, 可能被发现。还有其他方法可以发现新的 DNA 相互作用的蛋白质7, 但大多数依赖于类似芯片的方法8,9,10, 质粒插入11,12, 13、14或具有高拷贝数15的区域。相比之下, HyCCAPP 可以应用于多和单拷贝区域, 并且不需要任何先前关于该区域蛋白质的信息。此外, 虽然上面提到的一些方法有很有价值的特点, 特别是避免了 DNA 蛋白交联反应的需要, 但 HyCCAPP 的独特特点是它可以应用于未修饰细胞中的单拷贝区域, 而且没有任何事先了解假定的结合蛋白, 或可用的抗体。
在这一点上, HyCCAPP 主要用于分析酵母中的不同基因组区域4,5,6, 最近被用于分析α卫星 dna 中的蛋白质-DNA 相互作用, 重复区域在人类基因组16中。作为我们正在进行的工作的一部分, 我们已经适应了最初开发的酵母染色质的杂交捕获方法, 适用于人体细胞的分析, 并提出了一个修改的协议, 允许选择性捕获单拷贝目标人类基因组中的区域, 其效率与我们在酵母中的初步研究相似。这种新的优化协议现在允许技术的适应和利用, 通过质谱或其他分析方法来审问人类基因组中的蛋白质-DNA 相互作用。
重要的是要强调, HyCCAPP 方法是为了分析特定的目标区域, 还不适合于全基因组的分析。这项技术在处理那些缺乏相互作用的蛋白质信息的区域时尤其有用, 或者当需要对特定基因组轨迹上的相互作用蛋白质进行更全面的深入分析时。HyCCAPP 的目的是揭示 dna 结合蛋白, 但不能准确描述基因组 DNA 中特定的蛋白质结合点。在目前的实施中, 该方法并没有提供有关单个蛋白质的 DNA 结合序列或图案的信息。因此, 它很好地补充了现有的技术, 如自由放任, 并可能允许识别新的结合蛋白在基因组区域由初步的自由放任分析确定。

<table>
	<tbody>
		<tr>
			<td>PrimerQuest tool</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OligoAnalyzer 3.1</td>
			<td>IDT</td>
			<td></td>
			<td>Analyze capture oligonucleotids</td>
		</tr>
		<tr>
			<td>PairFold</td>
			<td>UBC</td>
			<td></td>
			<td>Analyze interactions</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Thermo Fisher</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>850 cm2 roller bottle&#160;</td>
			<td>Greiner Bio-one</td>
			<td>680058</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller system</td>
			<td>Wheaton</td>
			<td>22-288-525</td>
			<td></td>
		</tr>
		<tr>
			<td>37 % formaldehyde&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P4380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson digital sonifier SFX 150</td>
			<td>Emerson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0 fluorometer</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR assay kit</td>
			<td>Thermo Fisher</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 kit</td>
			<td>Agilent</td>
			<td>5067-1504</td>
			<td></td>
		</tr>
		<tr>
			<td>MES sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>M3885</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M</td>
			<td>Thermo Fisher</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Thermo Fisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Thermo Fisher</td>
			<td>12301D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 atreptavidin</td>
			<td>Thermo Fisher</td>
			<td>60210</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding tubes</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>SciGene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube shaker and rotator</td>
			<td>Thermo Fisher</td>
			<td>415110Q</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (RNase-free)</td>
			<td>New England BioLabs</td>
			<td>M0303</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC buffer 20&#215; concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>S6639</td>
			<td></td>
		</tr>
	</tbody>
</table>

adaptation of hybridization capture of chromatin-associated proteins for proteomics to mammalian cells

初步开发了用于蛋白质组学 (HyCCAPP) 技术的染色质相关蛋白的杂交捕获, 以揭示酵母中新的 DNA-蛋白质相互作用。它允许对目标区域进行分析, 而不需要事先了解绑定到目标区域的可能蛋白质。理论上, 这使得 HyCCAPP 可以用来分析任何感兴趣的基因组区域, 并且它提供了足够的灵活性, 可以在不同的细胞系统中工作。这种方法并不意味着研究已知转录因子的结合点, 这项任务更适合于染色质免疫沉淀 (芯片) 和类似芯片的方法。HyCCAPP 的力量在于它探索 DNA 区域的能力, 因为它有有限的或不了解与之相关的蛋白质。它也可以是一种方便的方法, 以避免偏见 (目前在芯片样的方法) 引入蛋白质的染色质浓缩使用抗体。潜在的, HyCCAPP 可以是一个强大的工具, 发现真正的新的 DNA 蛋白相互作用。迄今为止, 该技术主要应用于酵母细胞或哺乳动物细胞中的高复制重复序列。为了成为我们设想的强大工具, HyCCAPP 方法需要进行优化, 以有效地捕获哺乳动物细胞中的单拷贝基因座。在这里, 我们提出了我们对人类细胞系的初始酵母 HyCCAPP 捕获协议的适应, 并表明, 单拷贝染色质区域可以有效地与此修改的协议隔离。

由于需要大量的 HyCCAPP 的染色质来获得成功, 细胞生长到相对较高的融合水平。台盼蓝染色用于确认细胞死亡率适中 (< 10%)。在单拷贝实验中, 杂交捕获之前的染色质含量需要在 femtomolar 范围内, 通常需要至少10个9细胞作为起始材料。在进行全面实验之前, 建议在较小的批次中测试捕获寡核苷酸的性能。在图 1中显示了使用寡核苷酸滴定的杂交捕获。捕获寡核苷酸的数量逐渐增加, 而总浓度保持不变。这个实验清楚地显示了三关键方面。首先, 它有助于确定, 对于该特定区域, 需要多少寡核苷酸达到最佳 (和最大) 杂交效率。其次, 它揭示了在特定的寡核苷酸之间是否存在明显的有害作用。最后, 它表明, 是否有任何寡核苷酸具有很大的背景 (在这种情况下, 通过 qPCR 分析, 以引物针对其他区域的基因组) 的特异性较差。
由于染色质样品的交联性质, 捕获杂交的产量将显得温和。交联染色质中的大部分片段不适于杂交捕获。串联杂交实验证明了这一点 (图 2)。如果杂交捕获成功运行, 则使用不同捕获寡核苷酸的同一染色质材料的后续捕获实验将导致比使用新鲜染色质 (~ 11% 更低) 的产量 (略低于平均值)。但是, 如果同一区域再次瞄准同一组寡核苷酸在第二捕获实验中使用的染色质样本剩余的第一次捕获实验后, 产量将下降约 90%, 确认大多数的杂交材料已被捕获。如果在随后捕获同一组寡核苷酸之后没有观察到捕获的减少, 则表明杂交捕获步骤中存在技术问题。
在像酵母这样的简单系统中, 我们观察到 4%5的捕获效率, 这导致在不同条件下酵母生长时, 在2个区域发现9种蛋白质差异丰富。然而在人类细胞中, 基因组比酵母大250倍, 杂交产量通常低于 1% (图 3)。如图 3所示, 单个实验可能产生的蛋白质组分析的数量不足。在这种情况下, 几个捕获的样本将不得不汇集起来, 以便进行随后的质谱分析。或者, 较低浓度的捕获靶区染色质可用于靶向分析方法, 如西方印迹和质谱的选择反应监测化验。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57140/57140fig1.jpg"> 图 1: 捕获寡核苷酸滴定的方块图表示形式.通过 qPCR 对越来越多的捕获寡核苷酸进行了测试和分析。背景是指针对基因组中其他区域的 qPCR 化验。值被显示为从杂交捕获使用单一寡核苷酸的褶皱变化。胡须代表最小和最大值。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57140/57140fig1large.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/57140/57140fig2.jpg"> 图 2: 串行捕获的框图表示形式.序列杂交捕获与同一组寡核苷酸 (A_A) 显示一个强大的下降, 与序列杂交捕获使用不同的一组寡核苷酸 (A_B) (p = 3.88 e-5)。浓缩是用 qPCR 测量的, 而值是相对于用新鲜的染色质捕捉来显示的。胡须代表最小和最大值。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57140/57140fig2large.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/57140/57140fig3.jpg"> 图 3: 使用人类细胞进行杂交捕获.不同染色体区域的杂交捕获。每个区域都是另一个负面控制。此外, 使用被炒 (Scr) 序列作为真正的负控制来执行杂交捕获。误差线表示标准偏差。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57140/57140fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells at JoVE.com

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

这是一种方法, 以确定新的 DNA 相互作用的蛋白质在特定的靶点, 依赖序列特定捕获的交联染色质的后续蛋白质组分析。不需要事先了解潜在的结合蛋白, 也无需进行细胞修饰。该技术最初是为酵母开发的, 现在已经适应了哺乳动物细胞。

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

adaptation-hybridization-capture-chromatin-associated-proteins-for

Nanyang Technological University

Research

JoVE Journal

Genetics

6.2K 次观看.  Texas Biomedical Research Institute. 这是一种方法, 以确定新的 DNA 相互作用的蛋白质在特定的靶点, 依赖序列特定捕获的交联染色质的后续蛋白质组分析。不需要事先了解潜在的结合蛋白, 也无需进行细胞修饰。该技术最初是为酵母开发的, 现在已经适应了哺乳动物细胞。

视频: Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

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 ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

哺乳动物系统 CRISPR 指南 RNA 克隆

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...