Name: identification of footprints of rna:protein complexes via rna immunoprecipitation in tandem followed by sequencing (ripit-seq)
Uploaded: 2019-07-10T14:30:00
Description: Watch this Scientific Journal Video about Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq at JoVE.com

这项工作得到了NIH赠款GM120209(GS)的支持。作者感谢OSUCCC基因组共享资源核心的服务(CCC支持赠款NCI P30 CA16058)。

1. 建立稳定的 HEK293 细胞系,表达四环素诱导 FLAG 标记感兴趣的蛋白质 (POI)
<ol><li>种子 HEK293 细胞具有一个稳定增长的 Flp 重组靶 (FRT) 位点,在生长培养基的密度为 10 x 104细胞/mL(Dulbeco 的改性 Eagle 的培养基 [DMEM] = 10% 胎儿牛血清 [FBS] = 1% 青霉素-链球菌素 [penn/链球菌]井板。允许细胞在37°C和5%CO2(所有后续步骤的标准生长条件)的加湿培养箱中一夜之间生长。 </li>
	<li>第二天,细胞应为+70...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+challenges+in+the+detection+of+transcriptome&#8208;wide+protein-RNA+interactions.">Advances and challenges in the detection of transcriptome&#8208;wide protein-RNA interactions.</a> Wiley Interdisciplinary Reviews: RNA. 9 (1), (2018).</li><li>Singh, G., 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=The+Cellular+EJC+Interactome+Reveals+Higher-Order+mRNP+Structure+and+an+EJC-SR+Protein+Nexus.">The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus.</a> Cell. 151, 750-764 (2012).</li><li>Singh, G., Ricci, E. P., Moore, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIPiT-Seq:+A+high-throughput+approach+for+footprinting+RNA:protein+complexes.">RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes.</a> Methods. 65, 320-332 (2014).</li><li>Mabin, J. 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=The+Exon+Junction+Complex+Undergoes+a+Compositional+Switch+that+Alters+mRNP+Structure+and+Nonsense-Mediated+mRNA+Decay+Activity.">The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity.</a> Cell Reports. 25 (9), 2431-2446 (2018).</li><li>Gehring, N. H., Lamprinaki, S., Kulozik, A. E., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+Exon+Junction+Complexes+by+PYM.">Disassembly of Exon Junction Complexes by PYM.</a> Cell. 137 (3), 536-548 (2009).</li><li>Dostie, J., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+Is+Required+to+Remove+Y14+from+mRNAs+in+the+Cytoplasm.">Translation Is Required to Remove Y14 from mRNAs in the Cytoplasm.</a> Current Biology. 12 (13), 1060-1067 (2002).</li><li>Z&#252;nd, D., Gruber, A. R., Zavolan, M., M&#252;hlemann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation-dependent+displacement+of+UPF1+from+coding+sequences+causes+its+enrichment+in+3'+UTRs.">Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs.</a> Nature Structural &amp; Molecular Biology. 20 (8), 936-943 (2013).</li><li>Gangras, P., Dayeh, D. M., Mabin, J. W., Nakanishi, K., Singh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Identification+of+Recombinant+Argonaute-Bound+Small+RNAs+Using+Next-Generation+Sequencing.">Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.</a> Argonaute Proteins: Methods and Protocols. 1680, 1-28 (2018).</li><li>Heyer, E. E., Ozadam, H., Ricci, E. P., Cenik, C., Moore, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+kit-free+method+for+making+strand-specific+deep+sequencing+libraries+from+RNA+fragments.">An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments.</a> Nucleic Acids Research. 43 (1), 2 (2015).</li><li>Cameron, V., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3'-Phosphatase+activity+in+T4+polynucleotide+kinase.">3'-Phosphatase activity in T4 polynucleotide kinase.</a> Biochemistry. 16 (23), 5120-5126 (1977).</li><li>Ricci, E. P., 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=Staufen1+senses+overall+transcript+secondary+structure+to+regulate+translation.">Staufen1 senses overall transcript secondary structure to regulate translation.</a> Nature Structural &amp; Molecular Biology. 21 (1), 26-35 (2014).</li><li>Lackner, D. H., 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+generic+strategy+for+CRISPR-Cas9-mediated+gene+tagging.">A generic strategy for CRISPR-Cas9-mediated gene tagging.</a> Nature Communications. 6, 10237 (2015).</li><li>Metkar, 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=Higher-Order+Organization+Principles+of+Pre-translational+mRNPs.">Higher-Order Organization Principles of Pre-translational mRNPs.</a> Molecular Cell. 72 (4), 715-726 (2018).</li><li>Giudice, G., S&#225;nchez-Cabo, F., Torroja, C., Lara-Pezzi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATtRACT-a+database+of+RNA-binding+proteins+and+associated+motifs.">ATtRACT-a database of RNA-binding proteins and associated motifs.</a> Database: The Journal of Biological Databases and Curation. 2016, (2016).</li><li>Paz, I., Kosti, I., Ares, M., Cline, M., Mandel-Gutfreund, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBPmap:+a+web+server+for+mapping+binding+sites+of+RNA-binding+proteins.">RBPmap: a web server for mapping binding sites of RNA-binding proteins.</a> Nucleic Acids Research. 42, 361-367 (2014).</li><li>Sundararaman, 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=Resources+for+the+Comprehensive+Discovery+of+Functional+RNA+Elements.">Resources for the Comprehensive Discovery of Functional RNA Elements.</a> Molecular Cell. 61 (6), 903-913 (2016).</li></ol>

在这里,我们将讨论一些成功执行 RIPiT 的关键注意事项。首先,必须优化单个 IP,以便在每个步骤中实现尽可能高的效率。此处描述的输入细胞的FLAG agarose 珠子的量已被证明对于我们测试过的各种蛋白质是可靠的。由于只有一小部分伙伴蛋白与 FLAG 蛋白共同免疫,因此高效第二 IP 所需的抗体量通常较低(小于 10 μg)。小规模的RIPiT(从一个10厘米的板)然后西方印版验证蛋白质在每个部分在两个免疫沉淀步...

在细胞内,RNA与蛋白质复合体存在,形成RNA:蛋白质复合物(RNPs)。RNPs围绕RNA结合蛋白(RBPs,那些直接结合RNA的蛋白质)进行组装,但也包括非限制性核糖核酸(那些结合RPS但非RNA的),并且在性质上通常是动态的。RP 及其辅助因素在 RnP 中共同发挥作用,以执行监管职能。例如,在无意义介导的mRNA衰变(NMD)通路中,UPF蛋白(UPF1、UPF2和UPF3b)识别过早终止的核糖体。每个UPF蛋白都可以与RNA结合,但只有当它们聚集在一起时,活性NMD复合物才会开始形成。在这个复合体中,UPF1通过非RBP SMG1进一步通过磷酸化激活,这种UPF1激活最终导致mRNA衰变诱导因子1的招募,2。在此示例中,RBP 需要非 RBP 辅助因素来招募和激活触发 NMD 的 RNP 复合物。然而,RnPs的另一个属性是它们的组成异质性。考虑存在于不同E、A、B或C复合物中的拼接体。不同的拼接体复合物具有重叠和不同的蛋白质3。为了研究RNP功能,必须阐明哪些RNA被RBP及其相关蛋白质所束缚。有许多方法可以实现它,每种方法都有其明显的优点和缺点4,5,6,7。
广泛流行的方法来识别RBP结合位点 - 交联后免疫沉淀(CLIP)及其各种变异 - 依靠紫外线(UV)光将RBP与RNA8交联。然而,对于RNP中不直接接触RNA的非RRNA,这不是一种有效的方法。在这里,我们描述了一种适用于RSP和非限制性商业惯例的替代方法,用于分离和识别其RNA结合位点。这种方法称为RNA免疫沉淀串联(RIPiT)由两个免疫沉淀步骤组成,与单个纯化相比,这有助于实现更高的特异性(图1)9,10。与 CLIP 相比,单个免疫沉淀 (IP) 步骤可以以较低的严格性执行,因此 RIPiT 不依赖于在免疫沉淀期间能够承受强洗涤剂存在的抗体的可用性。RIPiT最独特的优势是能够以两个不同的纯化步骤靶向两种不同的蛋白质;这提供了一个强大的方法来丰富一个成分独特的RNP复合物从其他类似的复合物11。
对 RIPiT 程序的微小变化可以进一步增强 RNP 富集性。例如,RNA-蛋白质或蛋白质-蛋白质-蛋白质在RNPs内的一些相互作用是暂时的,可能难以有效地纯化这种复合物的足迹。为了稳定这种相互作用,在细胞裂解和RIPiT之前,RNPs可以在细胞内与甲醛交联。例如,我们观察到,外子结复合体 (EJC) 核心因子 EIF4AIII 和 EJC 拆解因子之间的弱相互作用,PYM12可以通过甲醛处理来稳定,以便更多 RNA 足迹得到丰富(数据不如图所示)。在细胞收获和RIPiT之前,细胞也可以用药物治疗,以稳定或丰富特定状态的RNP。例如,在研究在翻译过程中从mRNA中去除的蛋白质(例如,EJC 13,UPF114)时,使用翻译抑制剂(如紫霉素、环霉素或哈林托宁)进行治疗可导致增加的占用率。RNA上的蛋白质。
从RIPiT中恢复的RNA量通常较低(0.5-10 pmoles,即10-250 ng RNA,考虑到平均RNA长度为75 nt)。主要原因是,只有一小部分特定蛋白质与RNPs内的其他蛋白质复合存在(第一步中任何"自由"蛋白IP在第二个IP期间丢失)。为了从这个RNA生成RNA-Seq库,我们在此还概述了以前发布的协议,适用于这种低RNA输入15,16(图2),在3中产生高通量测序就绪样品。天。

<table border="0" cellpadding="0" cellspacing="0" style="width:1167px;" width="1166">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Anti-FLAG Affinity Gel</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:101px;">A2220</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">ATP, [&#947;-32P]- 3000Ci/mmol 10mCi/ml EasyTide, 250&#181;Ci</td>
			<td style="width:112px;">PerkinElmer</td>
			<td style="width:101px;">BLU502A250UC</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Disposable Syringes with Luer-Lok Tips (200)</td>
			<td style="width:112px;">Fisher</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Betaine 5M</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:101px;">B0300</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">biotin-dATP</td>
			<td style="width:112px;">TriLink</td>
			<td style="width:101px;">N-5002</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">biotin-dCTP</td>
			<td style="width:112px;">Perkin Elmer</td>
			<td style="width:101px;">NEL540001EA</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Branson Sonifier, Model SSE-1</td>
			<td style="width:112px;">Branson</td>
			<td style="width:101px;"></td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">CircLigase I</td>
			<td style="width:112px;">VWR</td>
			<td style="width:101px;">76081-606</td>
			<td style="width:591px;">ssDNA ligase I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">DMEM, High Glucose</td>
			<td style="width:112px;">ThermoFisher</td>
			<td style="width:101px;">11995-065</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">DNA load buffer NEB</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;"></td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Dynabeads Protein A</td>
			<td style="width:112px;">LifeTech</td>
			<td style="width:101px;">10002D</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Flp-In-T-REx 293 Cell Line</td>
			<td style="width:112px;">ThermoFisher</td>
			<td style="width:101px;">R78007</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">GeneRuler Low Range DNA Ladder</td>
			<td style="width:112px;">ThermoScientific</td>
			<td style="width:101px;">FERSM1203</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Hygromycin B</td>
			<td style="width:112px;">ThermoFisher</td>
			<td align="right" style="width:101px;">10687010</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mini-PROTEAN TBE Gel 10 well</td>
			<td style="width:112px;">Bio-Rad</td>
			<td align="right" style="width:101px;">4565013</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Mini-PROTEAN TBE-Urea Gel</td>
			<td style="width:112px;">Bio-Rad</td>
			<td align="right" style="width:101px;">4566033</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miRCAT-33 adapter 5&#8242;-TGGAATTCTCGGGTGCCAAGGddC-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mirus transIT-X2 transfection reagent</td>
			<td style="width:112px;">Mirus</td>
			<td style="width:101px;">MIR 6004</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mth RNA ligase</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;">E2610S</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE1.0 5&#8242;-AATGATACGGCGACCACCGAGATCTACACT 
			CTTTCCCTACACGACGCTCTTCCGATC*T-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE2.0 5&#8242;-CAAGCAGAAGACGGCATACGAGATCGGTCTC 
			GGCATTCCTGCTGAACCGCTCTTCCGATC*T-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">Phenol/Chloroform/Isoamyl Alcohol (25:24:1, pH 6.7, 100ml)</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:101px;">BP1752I-100</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Purple Gel Loading Dye (6x)</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;">NEB #7025</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Q5 DNA Polymerase</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;">M0491S/L</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:363px;">RNase I, E. coli, 1000 units</td>
			<td style="width:112px;">Eppicenter</td>
			<td style="width:101px;">N6901K</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">SPIN-X column</td>
			<td style="width:112px;">Corning</td>
			<td style="width:101px;">CLS8160-24EA</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Streptavidin beads</td>
			<td style="width:112px;">ThermoFisher</td>
			<td style="width:101px;">60210</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">Superscript III (SSIII)</td>
			<td style="width:112px;">ThermoScientific</td>
			<td align="right" style="width:101px;">18080044</td>
			<td style="width:591px;">reverse transcriptase enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SybrGold</td>
			<td style="width:112px;">ThermoFisher</td>
			<td style="width:101px;">S11494</td>
			<td style="width:591px;">gold nucleic acid gel stain</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:363px;">T4 Polynucleotide Kinase-2500U</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;">M0201L</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">T4RNL2 Tr. K227Q</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;">M0351S</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Tetracycline</td>
			<td style="width:112px;">Sigma</td>
			<td align="right" style="width:101px;">87128</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Thermostable 5&#180; App DNA/RNA Ligase</td>
			<td style="width:112px;">NEB</td>
			<td style="width:101px;">M0319S</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE1 5&#8242;-pGGCACTANNNNNAGATCGGAAGA 
			GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC 
			TTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE10 5&#8242;-pGGTGTTCNNNNNAGATCGGAAG 
			AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT 
			CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE11 5&#8242;-pGGTAAGTNNNNNAGATCGGAA 
			GAGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC 
			TTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE12 5&#8242;-pGGAGATGNNNNNAGATCGGAAGA 
			GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC 
			TTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE2 5&#8242;-pGGGTAGCNNNNNAGATCGGAAGAG 
			CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT 
			CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE35&#8242;-pGGTCGATNNNNNAGATCGGAAG 
			AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT 
			CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE4 5&#8242;-pGGCCTCGNNNNNAGATCGGAAGA 
			GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC 
			TTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE5 5&#8242;-pGGTGACANNNNNAGATCGGAAGA 
			GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC 
			TTCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE6 5&#8242;-pGGTAGACNNNNNAGATCGGAAGAG 
			CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC 
			CGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE7 5&#8242;-pGGGCCCTNNNNNAGATCGGAAG 
			AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCT 
			TCCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE8 5&#8242;-pGGATCGGNNNNNAGATCGGAAGAG 
			CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTT 
			CCGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq_SE9 5&#8242;-pGGACTGANNNNNAGATCGGAAGAG 
			CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC 
			CGATCTCCTTGGCACCCGAGAATTCCA-3&#8242;</td>
			<td>Any</td>
			<td></td>
			<td style="width:591px;">this protocol is only compatible with the Illumina sequencing platform</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">Typhoon 5 Bimolecular Imager</td>
			<td style="width:112px;">GE Healthcare Life Science</td>
			<td align="right" style="width:101px;">29187191</td>
			<td style="width:591px;"></td>
		</tr>
	</tbody>
</table>

identification of footprints of rna:protein complexes via rna immunoprecipitation in tandem followed by sequencing (ripit-seq)

RNA免疫沉淀串联(RIPiT)是一种在RNA:蛋白(RNP)复合物内丰富一对蛋白质的RNA足迹的方法。RIPiT 采用两个纯化步骤。首先,标记的RNP亚单位的免疫沉淀后是轻度RNase消化和随后的非变性亲和洗脱。另一个RNP亚单位的第二次免疫沉淀允许扩充定义的复合物。在RNA和蛋白质变性后,RNA足迹被转换成高通量的DNA测序库。与更流行的紫外线 (UV) 交联后免疫沉淀 (CLIP) 方法以丰富 RBP 结合位点不同,RIPiT 是紫外线交联独立。因此,RIPiT可应用于RNA相互作用中存在的众多蛋白质,这些蛋白质对RNA调控至关重要,但不直接接触RNA或紫外线交联,与RNA接触不良。RIPiT 中的两个纯化步骤提供了另一个优势,用于识别感兴趣的蛋白质与另一个共同因子合作作用的结合位点。双重纯化策略也有助于通过限制背景来增强信号。在这里,我们提供了一个逐步的过程来执行RIPiT,并从分离的RNA足迹生成高通量测序库。我们还概述了 RIPiT 的优势和应用,并讨论了它的一些局限性。

成功的RIPiT将导致感兴趣的蛋白质和其他已知相互作用蛋白的免疫沉淀,并且不存在非相互作用的蛋白质。如图3 A所示,在RIPiT洗脱中检测到马戈和EIF4AIII,但HNRNPA1没有(通道6)。同时,通过自光成像(图3B)或生物分析仪(图3C)检测出与RNP复合物共同纯化的RNA足迹。Puromycin治疗有望增加EJC对RNA的占用率,?...

Watch this Scientific Journal Video about Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq at JoVE.com

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

在这里,我们提出一个协议,以丰富内源RNA结合位点或"足迹"的RNA:蛋白质(RNP)复合物从哺乳动物细胞。这种方法涉及RNP亚单位的两个免疫沉淀,因此被称为RNA免疫沉淀串联(RIPiT)。

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

RNA免疫沉淀，然后测序，或RIPiT-Seq，是一种确定RNA区域由一对特定RNA结合蛋白结合的方法。RIPIT 最独特的优势是它能够在两种纯化中定位两种不同的蛋白质。这为与其他复合物的组成不同RNA蛋白复合物的丰富提供了一种强有力的方法。此外，RIPiT-Seq是UV-交叉链接独立的，可以应用于许多蛋白质，对RNA采取行动，而无需直接结合它。虽然这种方法不直接延伸到治疗或诊断，RNA结合蛋白和RNA处理已经与几个疾病，如神经病变和癌症相关。RIPiT-Seq为研究与疾病相关的RNA结合蛋白的功能提供了一个工具。RIPiT-Seq 需要一个可以表达表位标记蛋白质的系统。它只在 HEK293 细胞中进行了测试，并且最易接受细胞培养。然而，现在，由于我们有能力使用CRISPR标记蛋白质，RIPiT-Seq可以适用于不同的现代系统。RIPiT的成功取决于两种有效的免疫沉淀，因此优化所用抗体的免疫沉淀条件至关重要。此外，使用RNA需要适当的预防措施，因此在RNA提取期间和之后应使用无RNase试剂。证明这个程序将是劳伦伍德沃德，一个研究生在我的实验室。首先用每15厘米板15毫升的冰镇PBS轻轻清洗单层细胞。然后刮掉细胞到30毫升PBS，并在50毫升锥形管中收集它们。在4摄氏度下以400克离心10分钟，将细胞进行离心，然后丢弃上一液。加入四毫升冰冷低氧解液缓冲液，并使用P1000移液器重新悬浮细胞。将酸盐转移到五毫升管中，在冰上孵育10分钟。将酸盐放在冰浴中，然后根据手稿指示进行声波。然后加入108微升的五摩尔氯化钠，将盐浓度调整为150毫摩尔。然后，在4摄氏度下将12，000克的利酸盐离心10分钟，并收集20微升的上光剂，用于西方的印迹分析。根据手稿方向预洗 FLAG agarose 珠子，并在五毫升管中将剩余的上一升珠涂抹到 750 微升珠上。用细胞提取物孵育 FLAG agarose 珠子，在四摄氏度下用温和混合孵育一到三个小时。孵育后，在4摄氏度下以400克离心对珠子进行一分钟，并收集20微升的上经剂，用于西印分析。丢弃剩余的上流液，将珠子重新用四毫升的等洗缓冲液中重新填充。再次将珠子取出，取出上一液。根据手稿方向在 750 微升等值缓冲液中稀释 RNase I，并将其添加到 FLAG agarose 珠子中。在四摄氏度下用温和的混合孵育混合物，然后通过离心将珠子颗粒入。收集20微升的上光剂进行西印分析，并丢弃其余的。然后，用前文所述的等洗缓冲液清洗珠子。接下来，在珠子上加入375微升洗脱缓冲液，在4摄氏度下轻轻摇动混合物一到两个小时，进行亲和力洗脱。孵育后，将珠子粒粒并收集15微升的洗脱等分，用于西部印迹分析。当亲和力洗脱进行时，执行磁珠抗体结合。首先在一毫升等值缓冲液中清洗50微升磁珠。将珠子重新填充在100微升的结合缓冲液中，并加入适当量的抗体。然后，在室温下孵育珠子至少10分钟。用结合缓冲液清洗磁珠两次，并在 375 微升的 RIPiT 稀释缓冲液中重新悬浮磁珠。将珠子储存在冰上，直到免疫沉淀。将 FLAG 亲和力洗脱应用于抗体耦合磁珠，并在四摄氏度下温和混合一至两小时。用磁铁捕获磁珠，并收集15微升上一升用于西方印迹分析。然后，用等值缓冲液清洗珠子七次。在100微升透明样品缓冲器中重新悬浮磁珠，在冰上孵化悬浮液10分钟，继续变性洗脱。在孵育过程中，定期轻拂珠子以重新暂停。捕获磁铁上的磁珠，并收集 15 微升洗脱用于西方印迹分析。将剩余的洗脱转移到新的1.5毫升管中。为了提取RNA，在RIPIT洗脱中加入320微升无RNase水和400微升苯酚-氯仿异酰醇。涡流混合物，然后以12，000克离心5分钟。将水相的350微升收集到一个单独的管中。将醋酸钠、氯化镁、糖原和乙醇加入采集的样品中，并在负20摄氏度下孵育混合物过夜。第二天，在12，000克的离心中对RNA进行颗粒，在4摄氏度下30分钟，用70%乙醇清洗。乙醇洗涤后，使用T4多核苷酸激酶（PNK）处理RNA，不含任何ATP，并按照手稿说明进行苯酚-氯仿纯化。将清洁的RNA重新在4.5微升无RNase水中。为了量化RNA产量，请根据手稿指示用PNK和32P标记RNA进行放射性标记。使用低范围DNA梯和20至40核苷酸合成寡糖的大小和数量标准。在 26%尿素-聚丙烯酰胺凝胶上解析标记的 RNA。根据手稿指示干燥凝胶，将凝胶暴露在磷屏上，直到检测到足够的信号。此技术已用于研究 exon 结复合体或 EJC 的相互作用。从RIPIT程序的每个主要步骤中纯化的蛋白质的西方印迹分析表明，纯化的EJC同时含有EIF4A3和MAGOH。在洗脱中未检测到负控制 HNRNPA1。RNA足迹信号的强度与RNA蛋白相互作用量相关。当在用紫霉素治疗的细胞上执行 RIPIT 时，观察到更强的足迹，这增加了RNA上的EJC占用率。为了确保 RIPIT 有效，通过西方污点测试 IP 的效率非常重要。在进行深度测序之前，评估RNA的量和质量也很重要。2018年，Singh实验室成功地利用这项技术净化了外型结复合物的两种组合不同变异，并识别了RNA结合模式。如果研究人员选择通过自扫描检测RNA足迹，应遵循所有适当的辐射安全程序和协议。

Research

JoVE Journal

Genetics

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

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

科研

遗传学

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Circular RNAs using RNA Sequencing

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

番茄根转化后与拉子托尼亚索拉纳卡鲁姆接种，直接遗传分析细菌性威尔特病

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

植物病原体分泌效应器中RNA沉默抑制器的筛选和鉴定

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

通过旗-生物素串联纯化通过交叉链接和免疫沉淀对蛋白质-RNA相互作用进行转录组-全过程分析

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

使用染色质免疫沉淀和高通量测序（ChIP-seq）发现沙门氏菌生物膜中的CsgD监管目标

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...