Name: sample preparation for mass spectrometry-based identification of rna-binding regions
Uploaded: 2017-09-28T14:00:00.000+00:00
Duration: 10 min 52 s
Description: Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

导流洞得到了塞尔学者计划,. 史密斯基金会 (C1404) 和3月的硬币基金会 (1-财政年度-15-344) 的支持。R01GM110174 和 nih R01AI118891 以及国防部授予 BC123187P1 的支持。刻录-T。得到了 NIH 培训补助金 T32GM008216 的支持。

1. mESCs 的文化和扩展 注意: 小鼠胚胎干细胞易于培养, 可以迅速扩展到生物化学实验所需的大量时间, 这得益于它们的快速循环。健康的 mESCs 每12小时两倍. 
<ol> <li> 将 mESCs 扩展到所需的糊板上的制介质 (如下所示) 在37和 #176 的组织培养箱中; C、5% CO 2 和 #62; 95% 湿度. 
	注: 3-5 的生物复制为 + 4 su 样品和4SU 控制应准备足够的板材。一个10厘米的板块 ( 即 5-1000万细胞) 对于每个生物复制来说都足够了. </li>
	<li> mESCs 的培养基由 Dulbecco 和 #39; s 修饰的鹰和 #39; s 培养基 (DMEM), 补充15% 胎牛血清 (FBS), 2 毫米 l-谷氨酰胺, 0.5x 青霉素/链霉素, 1x 非必需氨基酸, 100 和 #181; M β-基, 1000 U/毫升白血病抑制因子 (LIF), 3 和 #181; CHIR99021, 1, #181; PD0325901. </li>
	<li> 在传代 mESCs 之前, 准备所需的 10 cm 板数. 
	<ol> <li> 在水中添加4毫升的0.1% 明胶, 确保覆盖整个表面; 在室温下孵育10分钟; 去除明胶溶液. </li>
	</ol> </li> <li> 通道单元格在每个板上达到 ~ 1000万单元格, 通常在接种后 48 h. 
	注: 当 mESCs 生长在稠密的菌落中时, 不应允许板块达到 100% 70-100, 如观察到生长在单分子膜上的细胞系 ( 如 HEK293 或3T3 细胞)。制板应传代当〜50% 汇合, 并明确之前, 介质变成黄色。
	<ol> <li> 要分离单元格, 用高密度制培养基在1x 磷酸缓冲盐水 (PBS) 中添加2毫米的 EDTA, 然后在 DMEM 中加入0.05% 的胰蛋白酶, 并在5分钟 (37 和 #176; C, 5% CO 2 , 和 #62; 95%湿度). </li>
		<li> 从孵化箱和重细胞中取出板, 移数次上下剧烈的 DMEM. </li>
		<li> 使用例计数单元格并确保单元格形成一个单细胞悬浮液 ( 即 无簇), 然后在室温下将细胞混合物 500 x g 离心5分钟。在 1-2 毫升 DMEM 中去除上清和重细胞颗粒. </li>
		使用步骤1.3.1 中制备的明胶涂层板, <li> 在密度为1万的细胞/cm 2 制培养基 ( 即每 10 cm 板上的 ~ 80万细胞) 中接种细胞. </li>
	</ol> </li> </ol> 2。蛋白质与 #8211 的交联; 活细胞中的 rna 相互作用 注意: rna-蛋白质的激活介导的 ribonucleoside 模拟 4-thiouridine (4SU). 4SU 具有比内源更长的吸光度核苷酸和只能纳入 RNA;因此, 中间波长的 UVB 可用于选择性地将 RNA 与蛋白质交联, 25 , 26 。UVB 治疗 4 su 处理的细胞导致共价通道4含苏 RNA 和氨基酸, 并报告偏好 Tyr, 跨国激进党, 满足, 赖氨酸, 和胱氨酸 27 . 
如步骤1所述, <ol> <li> 将 mESCs 扩展到所需的10厘米板数. 
	注: 3-5 的生物复制为 + 4 su 样品和4SU 控制应准备足够的板材。一个10厘米的板块 ( 即 5-1000万细胞) 是足够的每一个生物复制. </li>
	<li> 在板材到达70-100 之前, 将4SU 直接添加到介质中, 最终浓度为500和 #181; m. 离开-4SU 控制菜未经处理. </li>
	<li> 摇板轻轻地将4SU 均匀地分布在整个介质中。将它们返回到相同的组织培养孵化器2小时 (37 和 #176; C, 5% CO 2 , 和 #62; 95% 湿度). </li>
	<li> 将板移至冰桶并丢弃介质. </li>
	<li> 用5毫升的 ice-cold PBS 轻轻冲洗盘子. 
	注意: 轻轻冲洗或细胞可能分离. </li>
	<li> 添加2毫升的 ice-cold PBS 和放置无盖板的交, 配备有 UVB 发光灯泡 (312 nm)。照射板的能量设置为 1 J/厘米 2 . 
	注意: 这是至关重要的塑料盖被删除或他们可能保护细胞的紫外线和防止交联. </li>
	<li> 使用单元升降机收集细胞, 并将其转移到锥形管中的 ice-cold PBS。离心机在 2000 x g 为 5 min, 4 和 #176; C. </li>
	<li> 用2毫米 EDTA 和0.2 毫米磺氟化物 (PMSF) 去除5毫升 ice-cold PBS 中的上清和重细胞颗粒. </li>
	<li> 使用例计数单元格。预期分别为10厘米和15厘米的 5-10 和 10-2000万细胞. </li>
	<li> 2000 x g 离心5分钟, 4 和 #176; C. 
	注: 颗粒状细胞可立即用于步骤3或它们可以在液氮中闪光冷冻, 并储存在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 3。细胞核的分离 注意: 细胞核被分离以去除细胞质蛋白, 并增加核蛋白的覆盖率。这一步可以取代其他形式的细胞分馏研究限制性商业惯例在不同的蜂窝舱室. 
<ol> <li> 准备冰冷缓冲器 A (10 毫米三盐酸 pH 7.9, 4 和 #176; C, 1.5 毫米氯化镁 2 , 10 毫米氯化钾)。使用至少2.5 毫升每1000万细胞 (在步骤2.9 中计算), 以提取. 
	注: 缓冲器 a (如500毫升瓶) 的库存可以提前准备, 并储存在4和 #176; C 为6月。
	<ol> <li> 在使用前, 将 0.5 mm 糖 (PMSF) 和 0.2 mm 的 ice-cold 缓冲区 A 的所需数量 (每1000万个单元格) 添加到2.5 毫升. </li>
	</ol> </li> <li> 每1000万单元格使用 2 mL 缓冲来清洗单元格。在4和 #176 旋转 2500 x g, 5 分钟; C. </li>
	<li> 丢弃上清, 添加缓冲 a 补充0.2% 的非离子, 变性洗涤剂, 如辛基-苯氧基双-乙醇 (见 材料表 ), 500 和 #181; L 每1000万细胞。在4和 #176 旋转5分钟; C. </li>
	<li> 2500 x g 离心5分钟. </li>
	<li> 清除上清液; 颗粒包括完整的无细胞质的细胞核. 
	注: 核可立即用于4步或闪速冷冻在液氮和储存在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 4。细胞核的裂解 注意: 交联的细胞核在质谱-相容的缓冲液中裂解释放蛋白质和蛋白质-RNA 复合物. 
<ol> <li> 添加裂解缓冲液 (9 米尿素、100毫米三盐酸 pH 值8.0、24和 #176; C)、200和 #181; L 每1000万单元格。几种使用1/8 和 #34 的剪切染色质; 探头50% 振幅设置为 5-十年代. </li>
	<li> 旋转 1.2万 x g 为5分钟, 并收集175和 #181; L 上清, 以避免颗粒. 
	注: 100 及 #181; 我将用于下一个 RBR ID 步骤和剩余的75和 #181; 裂解液的 l 可以储存在-80 和 #176; C 供以后使用和/或西部印迹分析. </li>
	<li> 通过布拉德福德化验或类似技术测量蛋白质浓度 28 。将不同样品中的蛋白质浓度稀释到1毫克/毫升或最低的样品浓度, 使用适当数量的裂解缓冲液. 
	注: 从 1000万 mESCs 的细胞核中, 预计100和 #160; 200 和 #181; 蛋白质 g. </li>
</ol> 5。胰蛋白酶消化 注意: 蛋白质被消化生成多肽 suitable 为自下而上质谱 (MS) 分析. 
<ol> <li> 在核裂解液 (5 毫米决赛) 中添加 "", 在 RT 中孵育1小时. </li>
	<li> 从新鲜的库存中添加碘 (14 mM 决赛); 在黑暗中孵育30分钟. </li>
	<li> 稀释5倍体积50毫米 NH 的裂解液 4 小贩 3 </li>
	<li> 添加胰蛋白酶 (和 #181; g 蛋白: 和 #181; g 胰蛋白酶 = 200: 1); 孵育在37和 #176; 一夜之间. </li>
</ol> 6。多肽的脱盐 <ol> <li> 为每个示例准备一个 custom-made 阶段提示. 
	<ol> <li> 6.1.1. 将固相萃取 C18 材料放入200和 #181 的底部; L 吸管尖. </li>
		<li> 添加50和 #181; 在 C18 盘的顶部 R3 反相树脂。在1.5 毫升离心管内, 将舞台尖端放在离心适配器内, 并将接头放在带适配器的地方. </li>
		<li> 添加100和 #181; L 100% 乙腈至提示洗涤。离心机在 1000 x g 为1分钟, 以去除溶剂. </li>
		<li> 平衡50和 #181; L 0.1% 乙酸酸 (TFA)。离心机在 1000 x g 为 1 min. </li>
	</ol> </li> <li> 将100% 甲酸添加到已消化的蛋白质样品中, 以降低 pH 值到 2-3. 
	注: 这应该需要〜5和 #181; L 每1毫升稀释的肽样品, 但 pH 值应测量, 并增加更多的甲酸, 如果需要. </li>
	<li> 将样本加载到 custom-made 阶段提示, 在 1000 x g 上离心1分钟, 直到所有的样本都经过提示. </li>
	<li> 50 和 #181 清洗绑定多肽; 0.1% TFA。离心机在 1000 x g 为 1 min. </li>
	<li> 洗肽通过加入50和 #181; L 洗脱缓冲液 (75% 乙腈, 0.025% TFA) 到阶段尖端和离心机在 1000 x g 为1分钟, 迫使洗脱缓冲器的小费. </li>
	<li> 使用 150 x g 和 1-3 帕斯卡的真空离心机为1小时烘干样品 
	注: 干燥多肽可储存在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 7。交联 rna 的去除 注意: 用核酸处理多肽以去除交联 rna. 
<ol> <li> 重50和 #181 中的颗粒; L 2x 核酸缓冲器 (100 mm NH 4 小贩 3 , 4 mm 氯化镁 2 ). </li>
	<li> 使用 280 nm 的吸光度测定肽浓度, 假定1毫克/毫升的肽为 A 280 1. 
	注: 预期浓度为1毫克/毫升. </li>
	<li> 将所有样品调整到1毫克/毫升或最低浓度使用2x 核酸缓冲. </li>
	<li> 准备50和 #181 的主组合; l H 2 O + 1 和 #181; 高纯度核酸 (#8805; 250 U) (参考材料 表 ) 每样. </li>
	<li> 取50和 #181; 多肽样品 l, 添加50和 #181; H 2 O + 核酸主组合. </li>
	<li> 孵育37和 #176; C 为1小时进行质谱分析. 
	注: 多肽现在已经准备好进行质谱分析。他们可以立即分析或存储在-80 和 #176; C 无限期。这是一个潜在的停止点. </li>
</ol> 8。纳米液相色谱、质谱和原始数据处理 注意: 由于4的 su 交联改变了肽的质量, 它们的离子在 LC-ms/毫秒中不依赖于交肽的强度,因此, 这似乎是减少了交联。这种减少的程度和一致性反映了每个肽 24 的蛋白质-RNA 交联程度. 
<ol> <li> 制备 hplc 缓冲器, 如下所示: 0.1% 甲酸在 hplc 级水中 (缓冲器 a); 0.1% 甲酸在 hplc 级乙腈 (缓冲 B). </li>
	<li> 如果有可用的纳米高效液相色谱, 请将纳米柱与以下规格连接: 内径75和 #181; m; 用 C18-AQ 颗粒填充; 长度 20-25 厘米. 
	注: 如果高效液相色谱可在较高流速下运行, 则使用适当的列大小表示流速。高流量色谱法降低灵敏度. </li>
	<li> 程序的 HPLC 法如下: 流速 250-300 nL/分钟; 梯度从2到28% 缓冲 b 在 120 min, 从28到 80% b 为下 5 min 和等 80% b 为 10 min. 
	注: 如果 HPLC 没有自动列平衡之前的样品加载, 启动程序与10分钟在 0% B 之前加载样本. </li>
	<li> 程序的 MS 采集方法执行数据相关的获取 (多哈回合) 作为标准的猎枪蛋白质组学实验。仪器的工作周期应该交替使用最强烈信号的串联 ms 扫描 (或 ms) 进行全 ms 扫描。使用蛋白质组学 MS 运行的最佳设置. 
	注意: 要取得自信的结果, 请使用能够在高分辨率模式下执行至少全 MS 扫描的仪器 ( 例如 orbitrap、飞行时间)。要精确量化, 请确保全 MS 扫描之间的间隔不超过3秒. 较长的工作周期可能会阻止提取离子谱的精确定义. </li>
	<li> 加载 1-2 和 #181; 将样品加到 hplc 柱上, 并按程序运行 hplc-质谱/ms 法。对于每个生物复制执行至少两个独立的运行, 并视为技术复制. </li>
	<li> 在 ms 之后, 将原始文件导入到蛋白质组软件包中, 通过萃取离子色谱识别 ms 光谱和肽的定量。我们推荐可以执行多个 MS 运行的色谱对齐的软件包 (请参见 材料表 ) 29 , 30 。
	<li> 如果该选项在软件套件中可用, 请启用和 #34; 在开始数据处理之前, 在运行和 #34 之间进行匹配. </li>
	<li> 分析完成后, 将多肽列表连同量化值一起导出. </li>
</ol>

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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=A+triple+helix+stabilizes+the+3'+ends+of+long+noncoding+RNAs+that+lack+poly(A)+tails.">A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.</a> Genes Dev. 26 (21), 2392-2407 (2012).</li><li>Kim, T. K., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectural+and+Functional+Commonalities+between+Enhancers+and+Promoters.">Architectural and Functional Commonalities between Enhancers and Promoters.</a> Cell. 162 (5), 948-959 (2015).</li><li>Brannan, K. W., 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=SONAR+Discovers+RNA-Binding+Proteins+from+Analysis+of+Large-Scale+Protein-Protein+Interactomes.">SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.</a> Mol Cell. 64 (2), 282-293 (2016).</li><li>Bonasio, R., 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=Interactions+with+RNA+direct+the+Polycomb+group+protein+SCML2+to+chromatin+where+it+represses+target+genes.">Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes.</a> Elife. 3, e02637 (2014).</li><li>Kaneko, S., 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=Interactions+between+JARID2+and+noncoding+RNAs+regulate+PRC2+recruitment+to+chromatin.">Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.</a> Mol Cell. 53 (2), 290-300 (2014).</li><li>Kaneko, S., 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=Phosphorylation+of+the+PRC2+component+Ezh2+is+cell+cycle-regulated+and+up-regulates+its+binding+to+ncRNA.">Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.</a> Genes Dev. 24 (23), 2615-2620 (2010).</li><li>Saldana-Meyer, R., 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=CTCF+regulates+the+human+p53+gene+through+direct+interaction+with+its+natural+antisense+transcript,+Wrap53.">CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.</a> Genes Dev. 28 (7), 723-734 (2014).</li><li>Castello, 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=Comprehensive+Identification+of+RNA-Binding+Domains+in+Human+Cells.">Comprehensive Identification of RNA-Binding Domains in Human Cells.</a> Mol Cell. 63 (4), 696-710 (2016).</li><li>He, C., 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=High-Resolution+Mapping+of+RNA-Binding+Regions+in+the+Nuclear+Proteome+of+Embryonic+Stem+Cells.">High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells.</a> Mol Cell. 64 (2), 416-430 (2016).</li><li>Favre, 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=4-Thiouridine+photosensitized+RNA-protein+crosslinking+in+mammalian+cells.">4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.</a> Biochem Biophys Res Commun. 141 (2), 847-854 (1986).</li><li>Hafner, M., 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=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. 141 (1), 129-141 (2010).</li><li>Favre, A., Saintome, C., Fourrey, J. L., Clivio, P., Laugaa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thionucleobases+as+intrinsic+photoaffinity+probes+of+nucleic+acid+structure+and+nucleic+acid-protein+interactions.">Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.</a> J Photochem Photobiol B. 42 (2), 109-124 (1998).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Tyanova, S., Temu, T., Cox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MaxQuant+computational+platform+for+mass+spectrometry-based+shotgun+proteomics.">The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.</a> Nat Protoc. 11 (12), 2301-2319 (2016).</li><li>MacLean, 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=Skyline:+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments.">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li><li>Kondo, Y., Oubridge, C., van Roon, A. M., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+human+U1+snRNP,+a+small+nuclear+ribonucleoprotein+particle,+reveals+the+mechanism+of+5'+splice+site+recognition.">Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.</a> Elife. 4, (2015).</li><li>Hafner, M., 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=PAR-CliP--a+method+to+identify+transcriptome-wide+the+binding+sites+of+RNA+binding+proteins.">PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.</a> J Vis Exp. (41), (2010).</li></ol>

作者没有什么可透露的。

我们描述一个详细的实验协议, 以执行 RBR ID 在 mESCs, 并在适当的修改, 在任何细胞, 可以纳入4SU 到 RNA。其他的细胞类型可能需要优化的方法, 以确保足够的信噪比。此外, 虽然本文所述的议定书侧重于核限制性商业惯例的审查, 但 RBR ID 技术应易于适应不同的细胞隔间, 如胞或特定的器, 使用不同的分馏战略.可能需要优化的参数包括4SU 浓度和并网时间以及 UV 交联的能量。这些参数对于确保 RNA 蛋白?...

RBR ID 方法的目的是确定新的 rna 结合蛋白 (限制性商业惯例) 和映射他们的 rna 结合的区域 (RBRs) 与肽水平的决议促进 rna 结合的突变体的设计和研究生物和生物化学蛋白质-RNA 相互作用的作用。
RNA 在生物分子中是独一无二的, 因为它既能充当携带遗传信息的信使, 也可以折叠成复杂的三维结构, 其生物化学功能更类似于蛋白质的12。越来越多的证据表明, 编码 rna (rna) 发挥重要作用的各种基因调控和表观遗传途径3,4,5和, 通常, 这些管理功能是调解的演唱会与特定的 RNA 相互作用的蛋白质。特别重要的是, 一组相互作用的蛋白质最近被确定为强烈研究的长 ncRNA (lncRNA) Xist, 提供有价值的洞察如何这 lncRNA 介导的女性细胞 X 染色体失活6,7 ,8。值得注意的是, 这些 Xist 相互作用的蛋白质中有几个不包含任何规范的 rna 绑定域9, 因此它们的 rna 绑定活动不能根据它们的主序列单独被预测为在硅片中。考虑到数以千计的 lncRNAs 在任何给定的单元格10中表达, 有理由认为它们中的许多可能通过与尚未被发现的 RNA 结合蛋白 (限制性商业惯例) 的相互作用而行动。因此, 确定这些新的限制性商业惯例的实验战略将大大促进解剖 rna 生物功能的任务。
以往试图识别限制性商业惯例的经验依赖于波利亚 + RNA 选择耦合质谱 (MS)11,12,13,14,15。虽然这些实验在假定的限制性商业惯例中添加了许多蛋白质, 但通过设计他们只能检测到 polyadenylated 转录的蛋白质。然而, 大多数小 rna 和许多 lncRNAs 不是 polyadenylated 的。16,在这些实验中, 17及其相互作用的蛋白质可能会被遗漏。最近的一项研究应用机器学习蛋白质-蛋白质 interactome 数据库, 以识别蛋白质, co-purified 与多个已知的限制性商业惯例, 并表明这些经常性的惯例合作伙伴更有可能拥有 RNA 绑定活动18。然而, 这种方法依赖于挖掘现有的大型交互数据库, 并且只能识别可在变性条件下使用已知的限制性商业惯例 co-purified 的蛋白质, 从而排除不溶、膜嵌入和缺乏蛋白质.
将蛋白质鉴定为善意的限制性惯例通常不会自动产生关于蛋白质-RNA 相互作用的生物和/或生物化学功能的信息。为了解决这一点, 通常需要确定相互作用中所涉及的蛋白质域和氨基酸残基, 以便特定的突变体可以被设计成在每一种新的限制性条件下测试 RNA 结合的功能19,20. 我们的小组和其他人以前的努力使用重组蛋白片段和删除突变体来识别 RNA 结合区域 (RBRs)19,20,21,22;然而, 这种方法是 labor-intensive 和不相容的高通量分析。最近, 一项研究描述了使用质谱仪23以高通量方式绘制 RNA 结合活动的实验策略;然而, 这种方法依赖于双波利亚 + RNA 的选择, 因此与上面描述的限制性惯例识别方法具有相同的限制。
我们开发了一种技术, 称为 rna 结合区域识别 (RBR ID), 利用蛋白质 rna 光和定量质谱法来鉴定蛋白质和蛋白质区域与 RNA 在活细胞中的相互作用, 而不做假设在 RNA 多状态, 因而包括限制性商业惯例对波利亚-rna24。此外, 这种方法完全依赖于交联, 对蛋白质的溶解度或可达性没有要求, 因此适于在细胞膜内 (例如核包络) 或不太可溶性的隔间映射 RNA 结合活动 (例如核矩阵)。我们描述了对小鼠胚胎干细胞的细胞核进行 RBR 的实验步骤 (mESCs), 但稍加修改, 本协议应适用于多种细胞类型, 只要它们能有效地将4SU 从培养中.

<table><tbody><tr><td>KnockOut DMEM</td><td>Fisher Scientific</td><td>10829018</td><td></td></tr><tr><td>Fetal bovine serum, qualified, US origin</td><td>Fisher Scientific</td><td>26140079</td><td></td></tr><tr><td>L-Glutamine solution 200 mM</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Sigma</td><td>P0781</td><td></td></tr><tr><td>MEM Non-essential Amino Acid Solution (100&amp;times;)</td><td>Sigma</td><td>M7145</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>ESGRO Leukemia Inhibitory Factor (LIF)</td><td>EMD Millipore</td><td>ESG1106</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>PD0325901</td><td>Sigma</td><td>PZ0162</td><td></td></tr><tr><td>Gelatin solution,2% in water</td><td>Sigma</td><td>G1393</td><td></td></tr><tr><td>4-thiouridine</td><td>Sigma</td><td>T4509</td><td>50 mM stock in water</td></tr><tr><td>Spectrolinker XL-1500</td><td>Fisher Scientific</td><td>11-992-90</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma</td><td>78830</td><td></td></tr><tr><td>IGEPAL CO-630</td><td>Sigma</td><td>542334</td><td>Commercial form of octyl-phenoxy-polyethoxy-ethanol detergent</td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>I6125</td><td></td></tr><tr><td>Trypsin, sequencing grade</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Empore solid phase extraction disk</td><td>3M</td><td>66883</td><td></td></tr><tr><td>OLIGO R3 Reversed - Phase Resin</td><td>Fisher Scientific</td><td>1133903</td><td></td></tr><tr><td>Benzonase</td><td>Sigma</td><td>E8263</td><td>High purity nuclease</td></tr><tr><td>Sonic Dismembrator Model 100</td><td>Fisher Scientific</td><td>discontinued</td><td>updated with FB505110</td></tr><tr><td>HPLC grade acetonitrile</td><td>Fisher Chemical</td><td>A955-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W6 4</td><td></td></tr><tr><td>TFA</td><td>Fisher Scientific</td><td>A11650</td><td></td></tr><tr><td>Ammonium Bicarbonate</td><td>Sigma</td><td>A6141</td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma</td><td>49199</td><td></td></tr><tr><td>Formic Acid</td><td>Sigma</td><td>F0507</td><td></td></tr><tr><td>ReproSil-Pur 18-AQ</td><td>Dr. Maisch GmbH HPLC</td><td>r13.aq.0003</td><td>Packing material for HPLC column</td></tr><tr><td>Capillary for nano columns (75 &amp;micro;m)</td><td>Molex</td><td>1068150017</td><td></td></tr><tr><td>MaxQuant software</td><td>Max Planck Institute for Biochemistry</td><td></td><td>Can perform chromatographic alignment of multiple MS runs</td></tr></tbody></table>

sample preparation for mass spectrometry-based identification of rna-binding regions

编码 rna 在几个核过程中起重要作用, 包括调节基因表达、染色质结构和 DNA 修复。在大多数情况下, 编码 rna 的作用是由蛋白质介导的, 其功能反过来受这些相互作用与编码 rna。与此相一致的是, 越来越多的涉及核功能的蛋白质被报告为结合 rna, 在少数情况下, 这些蛋白质的 rna 结合区域已经被映射, 通常是通过费力的 candidate-based 方法。
在这里, 我们报告一个详细的协议, 以执行高通量, 蛋白质组范围内的 rna 结合蛋白和它们的 rna 结合区域的无偏见的识别。该方法依赖于在细胞 rna 中加入 photoreactive 苷模拟, 其次是紫外介导的蛋白质-rna 交联, 以及质谱分析, 以揭示蛋白质组内的 rna 交联肽。虽然我们描述了小鼠胚胎干细胞的过程, 但该协议应该很容易适应于各种培养细胞。

图 1描述了 RBR ID 工作流。由于这种技术的交联效率相对较低, 因此在生物复制中考虑到观察效果 (P-值) 的损耗水平和一致性非常重要。图 2显示了 RBR ID 结果的火山图。重叠 RNA 识别主题 (RRM) 域的多肽显示出高度一致的损耗水平。RRM 域可用作 RBR 分析的正则控制。
RBR ID 可用于...

Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

我们描述了一个协议, 以确定 rna 结合蛋白和地图他们的 rna 结合区域的活细胞使用紫外介导的光和质谱。

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

sample-preparation-for-mass-spectrometry-based-identification-rna

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.0K 次观看.  University of Pennsylvania Perelman School of Medicine. 我们描述了一个协议, 以确定 rna 结合蛋白和地图他们的 rna 结合区域的活细胞使用紫外介导的光和质谱。

视频: Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

生物化学

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

1H R1 + RNA 放松分散实验样品制备和设置的实用方面

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

生物学

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

RNA 结合区质量谱鉴定的样品制备

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rna 下拉法识别长非编码 rna 的 rna 靶点

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

¹H R_{1 +} RNA 放松分散实验样品制备和设置的实用方面

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

有针对性的RNA测序分析表征基因表达和基因组改变