Name: transcription start site mapping using super-low input carrier-cage
Uploaded: 2019-06-26T15:00:00
Duration: 6 min 59 s
Description: Watch this Scientific Journal Video about Transcription Start Site Mapping Using Super-low Input Carrier-CAGE at JoVE.com

这项工作得到了威尔康信托赠款(106954)的支持,该赠款授予了B.L.和医学研究委员会(MRC)核心基金(MC-A652-5QA10)。N.C.得到EMBO长期研究金的支持(EMBO ALTF 1279-2016);E. P. 得到了英国医学研究理事会的支持;B. L. 得到了英国医学研究理事会(MC UP 1102/1)的支持。

<ol>
	<li>Shiraki, T., 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=Cap+analysis+gene+expression+for+high-throughput+analysis+of+transcriptional+starting+point+and+identification+of+promoter+usage.">Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (26), 15776-15781 (2003).</li><li>Haberle, V., Lenhard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+architectures+and+developmental+gene+regulation.">Promoter architectures and developmental gene regulation.</a> Seminars in Cell and Developmental Biology. 57, 11-23 (2016).</li><li>Haberle, V., Stark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+core+promoters+and+the+functional+basis+of+transcription+initiation.">Eukaryotic core promoters and the functional basis of transcription initiation.</a> Nature Reviews Molecular Cell Biology. 19 (10), 621-637 (2018).</li><li>Andersson, 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=An+atlas+of+active+enhancers+across+human+cell+types+and+tissues.">An atlas of active enhancers across human cell types and tissues.</a> Nature. 507 (7493), 455-461 (2014).</li><li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Celniker, S. 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=Unlocking+the+secrets+of+the+genome.">Unlocking the secrets of the genome.</a> Nature. 459 (7249), 927-930 (2009).</li><li>Consortium, F., 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+promoter-level+mammalian+expression+atlas.">A promoter-level mammalian expression atlas.</a> Nature. 507 (7493), 462-470 (2014).</li><li>Boyd, 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=Characterization+of+the+enhancer+and+promoter+landscape+of+inflammatory+bowel+disease+from+human+colon+biopsies.">Characterization of the enhancer and promoter landscape of inflammatory bowel disease from human colon biopsies.</a> Nature Communications. 9 (1), 1661 (2018).</li><li>Adiconis, 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=Comprehensive+comparative+analysis+of+5'-end+RNA-sequencing+methods.">Comprehensive comparative analysis of 5'-end RNA-sequencing methods.</a> Nature Methods. , (2018).</li><li>Cvetesic, N., 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=SLIC-CAGE:+high-resolution+transcription+start+site+mapping+using+nanogram-levels+of+total+RNA.">SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA.</a> Genome Research. 28 (12), 1943-1956 (2018).</li><li>Murata, 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=Detecting+expressed+genes+using+CAGE.">Detecting expressed genes using CAGE.</a> Methods in Molecular Biology. 1164, 67-85 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9, 357 (2012).</li><li>A language and environment for statistical computing. Available from: <a target="_blank" href="https://www.R-project.org/">https://www.R-project.org/</a> (2017)</li><li>Haberle, V., Forrest, A. R., Hayashizaki, Y., Carninci, P., Lenhard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAGEr:+precise+TSS+data+retrieval+and+high-resolution+promoterome+mining+for+integrative+analyses.">CAGEr: precise TSS data retrieval and high-resolution promoterome mining for integrative analyses.</a> Nucleic Acids Research. 43 (8), e51 (2015).</li><li>Poulain, 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=NanoCAGE:+A+Method+for+the+Analysis+of+Coding+and+Noncoding+5'-Capped+Transcriptomes.">NanoCAGE: A Method for the Analysis of Coding and Noncoding 5'-Capped Transcriptomes.</a> Methods in Molecular Biology. 1543, 57-109 (2017).</li><li>Schon, M. A., Kellner, M. J., Plotnikova, A., Hofmann, F., Nodine, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoPARE:+parallel+analysis+of+RNA+5'+ends+from+low-input+RNA.">NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.</a> Genome Research. 28 (12), 1931-1942 (2018).</li></ol>

可降解载体RNA/DNA的专利已经填补。

要成功进行 SLIC-CAGE 库准备,使用低结合提示和管来防止样品吸附导致样品损失至关重要。在涉及检索上清液的所有步骤中,建议恢复整个样本体积。由于该协议具有多个步骤,连续样本丢失将导致库失败。
如果 CAGE (nAnT-iCAGE) 没有正常执行,最好用相同总RNA样本的不同输入量(10 ng、20 ng、50 ng、100 ng、200 ng)测试SLIC-CAGE,并与使用5μg总RNA制备的nAnT-iCAGE库进行比较。如果 nAnT-iCAGE 库不成功(小于每个样本获得的 DNA 库的 0.5-1 ng),SLIC-CAGE 不太可能工作,并且需要将样本损失降至最低。
确保没有未封顶的降解RNA或rRNA的高质量库是关键步骤,第7节所述的封顶。非常重要的是,链球菌素珠子在洗涤缓冲液中彻底悬浮,在继续下一步洗涤步骤或 cDNA 洗脱之前,清除洗涤缓冲液。
如果第一轮载波降级后qPCR的结果显示适配器_f1和载波_f1引能的使用没有区别,仍建议继续使用该协议。如果在第二轮载波降级后,Ct 值的差异小于 5,则建议进行第三轮载波降级。我们从来没有发现第三轮降解是必要的,如果发生,建议取代宿主内分糖种群。
如果获得的库的最终量不足以进行测序,则可在协议中添加其他多轮 PCR 扩增。然后,PCR 扩增可以设置所需的最小扩增周期,以产生足够的材料进行测序,同时考虑到在尺寸选择中无法避免的样品损失。然后,应使用 SPRI 磁珠进行纯化或尺寸选择,直到去除所有小 (<200 bp) 片段(如果需要,使用 0.6:1 珠与采样比),并且库应使用 Picogreen 进行量化。
库可以在单端或成对端模式下排序。使用成对端测序,可以获得有关转录等形的信息。此外,由于使用随机引物(TCT-N 6,N6是随机六项机)进行逆转录,因此,来自测序3'端的信息可用作唯一分子标识符(UMI),以折叠PCR重复项。由于使用适量的PCR扩增周期(最多18个),以前发现使用UMIs是不必要的。
由于协议的核心依赖于nAnT-iCAGE1,SLIC-CAGE使用8个条形码。因此,当前不支持多路复用超过 8 个示例。此外,SLIC-CAGE 和 nAnT-iCAGE 都不适合捕获小于 200 bp 的 RNA,因为协议旨在通过使用 AMPure XP 磁珠排除尺寸来去除链接器和 PCR 人工制品。
SLIC-CAGE 是唯一一种使用总RNA材料的纳米图绘制转录起始位点的无偏低输入单核苷酸分辨率方法。替代方法依赖于逆转录酶的模板切换活性,以条形码封盖RNA,而不是捕获上限(例如,NanoCAGE15和 NanoPARE16)。由于模板切换,这些方法在TSS检测中表现出序列特定的偏差,导致误报TS的数量减少和真TSS9、10的数量减少。

基因表达(CAGE)的帽分析是一种用于单核苷酸解析全基因组图谱的RNA聚合酶II转录起始位点(TSSs)1。其定量性质还允许 5' 端为中心的表达式分析。围绕TSS(约40 bp上游和下游)的区域是核心启动子,代表RNA聚合酶II和一般转录因子结合的物理位置(前2、3)。有关 TSS 的确切位置的信息可用于核心启动器发现和监测启动子动力学。此外,由于活性增强剂表现出双向转录的特征,CAGE数据也可用于增强剂发现和监测增强剂动力学4。CAGE方法最近越来越受欢迎,因为它在诸如ENCODE 5、modENCODE6和FANTOM项目7等备受瞩目的研究项目中有着广泛的应用和用途。此外,TSS 信息也被证明是重要的区分健康和疾病组织,因为疾病特异性的TSS可用于诊断目的8。
尽管有几种 TSS 映射方法可用(CAGE、RAMPAGE、STRT、纳米笼、纳米CAGE-XL、寡核封盖),但我们和其他人最近已经表明,CAGE 是最无偏见的方法来捕获假阳性数最少的真实 TSS9,10.最近的CAGE协议,nAnT-iCAGE11,是TSS分析最公正的协议,因为它避免使用限制性酶将片段切割成短标记,并且不使用PCR扩增。nAnT-iCAGE 协议的局限性是需要大量起始材料(例如,每个样品的总 RNA 量为 5 μg)。为了回答具体、与生物学相关的问题,通常无法获得如此大量的起始材料(例如,对于FACS排序的细胞或早期胚胎阶段)。最后,如果nAnT-iCAGE成功,每个样本只能获得1-2 ng的DNA库材料,从而限制了可实现的测序深度。
为了仅使用总RNA的纳米图进行TSS分析,我们最近开发了超低输入载波-CAGE 10(SLIC-CAGE,图1)。SLIC-CAGE 只需要总 RNA 的 10 纳克才能获得高复杂度库。我们的协议依赖于精心设计的合成RNA载体添加到感兴趣的RNA,实现总共5μg的RNA材料。合成载体在长度分布上模仿目标DNA库,以避免由过量的均质分子引起的潜在偏差。载体的序列基于大肠杆菌-tRNA合成酶基因(表1)的序列,原因有二。首先,最终库中载体的任何剩余,即使已测序,也不会映射到真核基因组。其次,由于大肠杆菌是一种嗜血菌物种,其内务管理基因针对适用于SLIC-CAGE的温度范围进行了优化。载体序列还嵌入了宿主内源内切酶识别位点,允许从载体RNA分子中提取的DNA进行特定降解。目标样本派生库保持不变,因为宿主内分酶识别站点很长(I-CeuI = 27 bp;I-SceI = 18 bp),在真核基因组中不太可能发现。在载体具体降解和通过大小排除去除碎片后,目标库被扩增,为下一代测序做好准备。根据起始RNA量(1-100纳克),预计在13-18PCR扩增周期之间。每个样本的最终DNA量在5-50 ng之间,产生足够的材料进行深度测序。当只使用总RNA的1-2纳克时,可以检测到真正的TSS;但是,这些库的复杂性预计会较低。最后,由于SLIC-CAGE基于nAnT-iCAGE协议11,因此在测序之前,它能对多达8个样本进行多路复用。

<table>
	<tbody>
		<tr>
			<td>2-propanol, Bioultra, for molecular biology, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>59304-100ML-F</td>
			<td>Used in RNAclean XP purification.</td>
		</tr>
		<tr>
			<td>3&#39; linkers</td>
			<td></td>
			<td></td>
			<td>Sequences are described in Murata et al 2014 and Supplementary Table 1 of this manuscript. Annealing of strands to produce 3&#39;linkers is described in the supplementary of this protocol.</td>
		</tr>
		<tr>
			<td>5&#39; linkers</td>
			<td></td>
			<td></td>
			<td>Sequences are described in Murata et al 2014 and Supplementary Table 1 of this manuscript. Annealing of strands to produce 5&#39;linkers is described in the supplementary of this protocol.</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP, 60 mL</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Purification of DNA</td>
		</tr>
		<tr>
			<td>Agencourt RNAClean XP Kit</td>
			<td>Beckman Coulter</td>
			<td>A63987</td>
			<td>Purification of RNA and RNA:cDNA hybrids in CAGE steps.</td>
		</tr>
		<tr>
			<td>Axygen 0.2 mL Polypropylene PCR Tube Strips and Domed Cap Strips</td>
			<td>Axygen (available through Corning)</td>
			<td>PCR-0208-CP-C</td>
			<td>Or any 8-tube PCR strips (used only for water and mixes).</td>
		</tr>
		<tr>
			<td>Axygen 1 x 8 strip domed PCR caps</td>
			<td>Axygen (available through Corning)</td>
			<td>PCR-02CP-C</td>
			<td>Caps for PCR plates.</td>
		</tr>
		<tr>
			<td>Axygen 1.5 mL Maxymum Recovery Snaplock Microcentrifuge Tube</td>
			<td>Axygen (available through Corning)</td>
			<td>MCT-150-L-C</td>
			<td>Low-binding 1.5 ml tubes, used for enzyme mixes or sample concentration.</td>
		</tr>
		<tr>
			<td>Axygen 96 well no skirt PCR microplate</td>
			<td>Axygen (available through Corning)</td>
			<td>PCR-96-C</td>
			<td>Low-binding PCR plates - have to be used for all steps in the protocol. Note that plates should be cut to contain 2 x 8 wells for easier visibility of the samples</td>
		</tr>
		<tr>
			<td>Bioanalyzer (or Tapestation): RNA nano and HS DNA kits</td>
			<td>Agilent</td>
			<td></td>
			<td>To determine quality of RNA, efficient size selection and final quality of the library (Tapestation can also be used)</td>
		</tr>
		<tr>
			<td>Biotin (Long Arm) Hydrazide</td>
			<td>Vector laboratories</td>
			<td>SP-1100</td>
			<td>Biotinylation/tagging</td>
		</tr>
		<tr>
			<td>Cutsmart buffer</td>
			<td>NEB</td>
			<td></td>
			<td>Restriction enzyme buffer</td>
		</tr>
		<tr>
			<td>Deep Vent (exo-) DNA Polymerase</td>
			<td>NEB</td>
			<td>M0259S</td>
			<td>Second strand synthesis</td>
		</tr>
		<tr>
			<td>DNA Ligation Kit, Mighty Mix</td>
			<td>Takara</td>
			<td>6023</td>
			<td>Used for 5&#39; and 3&#39;-linker ligation</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM each)</td>
			<td>ThermoFisher Scientific</td>
			<td>18427013</td>
			<td>dNTP mix for production of carrier templates (or any dNTPs suitable for PCR)</td>
		</tr>
		<tr>
			<td>Dynabeads M-270 Streptavidin</td>
			<td>Invitrogen</td>
			<td>65305</td>
			<td>Cap-trapping. Do not use other beads as these are optimised with the buffers used.</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12321D</td>
			<td>Magnetic stand for 1.5 ml tubes - used to prepare Streptavidin beads.</td>
		</tr>
		<tr>
			<td>DynaMag-96 Side Skirted Magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12027</td>
			<td>Magnetic stand for PCR plates (96 well-plates) - used with cut plates to contain 2 x 8 wells.</td>
		</tr>
		<tr>
			<td>Ethanol, BioUltra, for molecular biology, &#8805;99.8%</td>
			<td>Sigma-Aldrich</td>
			<td>51976-500ML-F</td>
			<td>Used in AMPure washes. Any molecular biology suitable ethanol can be used.</td>
		</tr>
		<tr>
			<td>Exonuclease I (E. coli)</td>
			<td>NEB</td>
			<td>M0293S</td>
			<td>Leftover primer degradation</td>
		</tr>
		<tr>
			<td>Gel Loading Dye, Purple (6x), no SDS</td>
			<td>NEB</td>
			<td>B7025S</td>
			<td>agarose gel loading dye</td>
		</tr>
		<tr>
			<td>HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td>New England Biolabs</td>
			<td>E2040S</td>
			<td>Kit for carrier in vitro transcription</td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis apparatus</td>
			<td></td>
			<td></td>
			<td>purification of carrier DNA templates from agarose gels</td>
		</tr>
		<tr>
			<td>I-Ceu</td>
			<td>NEB</td>
			<td>R0699S</td>
			<td>Homing endonuclease used for carrier degradation.</td>
		</tr>
		<tr>
			<td>I-SceI</td>
			<td>NEB</td>
			<td>R0694S</td>
			<td>Homing endonuclease used for carrier degradation.</td>
		</tr>
		<tr>
			<td>KAPA HiFi HS ReadyMix (2x)</td>
			<td>Kapa Biosystems (Supplied by Roche)</td>
			<td>KK2601</td>
			<td>PCR mix for target library amplification</td>
		</tr>
		<tr>
			<td>KAPA SYBR FAST qPCR kit (Universal) 2x</td>
			<td>Kapa Biosystems (Supplied by Roche)</td>
			<td>KK4600</td>
			<td>qPCR mix to assess degradation efficiency and requiered number of PCR amplification cycles</td>
		</tr>
		<tr>
			<td>Micropipettes and multichannel micropipettes (0.1-10 &#181;l, 1-20 &#181;l, 20-200 &#181;)</td>
			<td>Gilson</td>
			<td></td>
			<td>Use of Gilson with the low-binding Sorenson tips is recommended. Other micropippetes might not be compatible.. Different brand low-binding tips may not be of equal quality and may increase sample loss.</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td></td>
			<td></td>
			<td>For Picogreen concentration measurement of the final library. Microplates are used to allow small volume measurement and reduce sample waste.</td>
		</tr>
		<tr>
			<td>nuclease free water</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9937</td>
			<td>Or any nuclease (DNase and RNase) free water</td>
		</tr>
		<tr>
			<td>PCR thermal cycler</td>
			<td></td>
			<td></td>
			<td>incubation steps and PCR amplficication</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>F530S</td>
			<td>DNA polymerase for amplification of carrier templates (or any high fidelity polymerase)</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Purification of carrier PCR templates from agarose gels.</td>
		</tr>
		<tr>
			<td>qPCR machine</td>
			<td></td>
			<td></td>
			<td>determining PCR amplification cyle number and degree of carrier degradation</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>P11495</td>
			<td>Used to measure final library concentration - recommended as, in our hands, it is more accurate and reproducible than Qubit.</td>
		</tr>
		<tr>
			<td>Quick-Load Purple 100 bp DNA Ladder</td>
			<td>NEB</td>
			<td>N0551S</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>Quick-Load Purple 1 kb Plus DNA Ladder</td>
			<td>NEB</td>
			<td>N0550S</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>Ribonuclease H</td>
			<td>Takara</td>
			<td>2150A</td>
			<td>Digestion of RNA after cap-trapping.</td>
		</tr>
		<tr>
			<td>RNase ONE Ribonuclease</td>
			<td>Promega</td>
			<td>M4261</td>
			<td>Degradation of single stranded RNA not protected by cDNA.</td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>Removal of carrier DNA templates after in vitro transcription.</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>For cleanup of carrier RNA from in vitro transcription or capping</td>
		</tr>
		<tr>
			<td>Sodium acetate, 1 M, aq.soln, pH 4.5 RNAse free</td>
			<td>VWR</td>
			<td>AAJ63669-AK</td>
			<td>Or any nuclease (DNase and RNase) free solution</td>
		</tr>
		<tr>
			<td>Sodium acetate, 1 M, aq.soln, pH 6.0 RNAse free</td>
			<td></td>
			<td></td>
			<td>Or any nuclease (DNase and RNase) free solution</td>
		</tr>
		<tr>
			<td>Sodium periodate</td>
			<td>Sigma-Aldrich</td>
			<td>311448-100G</td>
			<td>Oxidation of vicinal diols</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MicroReach Guard, volume range 10 &#956;L, Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719390-960EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MultiGuard, volume range 1000 &#956;L , Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719463-1000EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MultiGuard, volume range 20 &#956;L , Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719412-960EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>Sorenson low binding aerosol barrier tips, MultiGuard, volume range 200 &#956;L , Graduated</td>
			<td>Sorenson (available through SIGMA-ALDRICH)</td>
			<td>Z719447-960EA</td>
			<td>Low-binding tips - recommended use throughout the protocol to minimise sample loss.</td>
		</tr>
		<tr>
			<td>SpeedVac Vacuum Concentrator</td>
			<td></td>
			<td></td>
			<td>concentrating samples in various steps to lower volume</td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080044</td>
			<td>Used for reverse transcription (1st CAGE step)</td>
		</tr>
		<tr>
			<td>Trehalose/sorbitol solution</td>
			<td></td>
			<td></td>
			<td>Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>Tris-HCl, 1M aq.soln, pH 8.5</td>
			<td></td>
			<td></td>
			<td>1 M solution, DNase and RNase free</td>
		</tr>
		<tr>
			<td>tRNA (20 mg/mL)</td>
			<td></td>
			<td></td>
			<td>tRNA solution. Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16520050</td>
			<td>Or any suitable pure low-melt agarose.</td>
		</tr>
		<tr>
			<td>USB Shrimp Alkaline Phosphatase (SAP)</td>
			<td>Applied Biosystems (Provided by ThermoFisher Scientific)</td>
			<td>78390500UN</td>
			<td></td>
		</tr>
		<tr>
			<td>USER Enzyme</td>
			<td>NEB</td>
			<td>M5505S</td>
			<td>Degradation of 3&#39;linker&#39;s upper strand, Uracil Specific Excision Reagent/Enzyme</td>
		</tr>
		<tr>
			<td>Vaccinia Capping System</td>
			<td>NEB</td>
			<td>M2080S</td>
			<td>Enzymatic kit for in vitro capping of carrier molecules</td>
		</tr>
		<tr>
			<td>Wash buffer A</td>
			<td></td>
			<td></td>
			<td>Cap trapping washes. Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>Wash buffer B</td>
			<td></td>
			<td></td>
			<td>Cap trapping washes. Preparation is described in Murata et al 2014.</td>
		</tr>
		<tr>
			<td>Wash buffer C</td>
			<td></td>
			<td></td>
			<td>Cap trapping washes. Preparation is described in Murata et al 2014.</td>
		</tr>
	</tbody>
</table>

transcription start site mapping using super-low input carrier-cage

基因表达(CAGE)帽分析是一种用于RNA聚合酶II转录起始位点(TSSs)的单核苷酸分辨率检测方法。准确检测 TSS 可增强核心启动子的识别和发现。此外,可以通过双向转录启动的签名来检测有源增强剂。此处介绍用于执行超低输入载波 CAGE(SLIC-CAGE)的协议。CAGE协议的这种SLIC适应通过人工增加RNA量,通过使用在感兴趣的样品中加入的体外转录RNA载体混合物,从而从纳米图总量中制备,从而最大限度地减少了RNA损失RNA(即数千个细胞)。载体模拟预期的DNA库片段长度分布,从而消除由同质载体丰度引起的偏差。在协议的最后阶段,载体通过带宿主内分酶的降解被移除,目标库被放大。目标样本库受到保护,防止降解,因为宿主内分酶识别位点很长(在18至27bp之间),使得它们在真核基因组中存在的概率非常低。最终结果是DNA库为下一代测序做好准备。协议中的所有步骤,至顺序,可在 6 天内完成。承运人准备需要一个完整的工作日;然而,它可以大量制备,并保持在-80°C冷冻。一旦测序,读取可以处理获得全基因组的单核苷酸分辨率TSS.TSS可用于核心促进剂或增强剂发现,提供对基因调控的洞察。聚合到启动器后,数据还可用于 5' 为中心的表达式分析。

本报告描述了从起始总RNA材料的纳米图中获取测序就绪库的完整SLIC-CAGE协议(图1)。为了获得合成RNA载体混合物,首先,PCR载体模板需要制备和凝胶纯化,以消除PCR侧产物(图2A)。每个PCR模板(共10个)是使用共同的正向,但不同的反向引物(表2),导致PCR模板的不同长度,使合成RNA载体的大小变异。一旦纯化,PCR模板用于载体分子的体外转录。如果模板经过凝胶纯化,则预期会提供单个RNA载体产品(参见图2B中的代表性凝胶分析)。载体的制备可根据需要进行升级,在制备时,混合和冷冻在-80°C下供将来使用。
使用推荐的最小样品总RNA(10 ng)结合16-18周期的PCR扩增,可以达到高复杂性SLIC-CAGE库。扩增最终库所需的PCR周期数在很大程度上取决于总输入RNA的数量(预期周期数在表4中)。
在第一轮降级后,在 qPCR 结果(步骤 17)中,使用适配器_f1 或载波_f1 引物获得的 Ct 值之间的预期差值为 1-2,使用适配器_f1 比载波_f1 低获得 Ct 值。
最终库中片段长度的分布在 200-2,000 bp 之间,平均片段大小为 700-900 bp(基于使用生物分析仪软件的区域分析,图 4B,D)。如图4A,C所示,较短的片段必须通过额外的大小排除(步骤20-21)来移除。这些短片段是 PCR 扩增人工制品,而不是目标库。请注意,较短的片段在测序流单元上更好地聚类,并可能导致排序问题。
每个样本获得库材料的预期量在 5-50 纳克之间。显著降低数量表明在协议期间样品损失。如果获得的低数量足以进行测序(需要 2-3 ng 的池库),则库的复杂性可能较低(见下文)。
根据测序机的不同,可能需要优化加载到流单元的库数量。使用 Illumina HiSeq 2500,加载 8-12 pM SLIC-CAGE 库平均提供 1.5 亿-2 亿次读取,其中 80% 的读取通过质量分数 Q30 作为阈值。
然后,获取的读取映射到参考基因组 [对于 50 bp 读取,Bowtie212可与允许每个种子序列(22 bp)零不匹配的默认参数一起使用]。预期的映射效率取决于总RNA输入量,如表5。然后,可以将唯一映射的读取加载到 R 图形和统计计算环境13中,并使用 CAGEr(生物导体封装14)进行处理。包晕影易于理解,并详细介绍了映射数据的工作流和处理。库复杂性的一个简单可视化控制是启动子宽度的分布,因为低复杂度库将人为地缩小启动子(图5A,SLIC-CAGE库派生自总RNA的1 ng,详情请见前文出版物10。然而,即使是低复杂度SLIC-CAGE库也能识别真正的CTSS,其精度比低/中位TSS映射的替代方法更精确(图5B,C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59805/59805fig1.jpg"> 图 1:SLIC-CAGE 协议中的步骤。样品RNA与RNA载体混合物混合,达到总RNA材料的5μg。cDNA通过逆转录合成,盖使用酸钠氧化。氧化允许使用生物素氢化物将生物素附着在帽子上。生物蛋白附着在mRNA的3°端,因为它也使用酸钠被氧化。为了从mRNA:cDNA杂交物中消除生物素,从未完全合成的cDNA和mRNA的3°端,用RNase I.cDNA处理,然后通过链球菌素磁珠的亲亲纯化选择达到mRNA5°末端的rNase I.cDNA(捕获帽)。释放cDNA后,5+和3+链接器被连接。源自载体的库分子使用I-SceI和I-CeuI致内分酶降解,并使用SPRI磁珠去除片段。然后,库被放大。<a href="https://www.jove.com/files/ftp_upload/59805/59805fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59805/59805fig2.jpg"> 图2:载体PCR模板和载体体外转录本的代表性凝胶分析。(A)凝胶纯化前的载体PCR模板:第一口井包含1 kbp标记,然后是载体PCR模板1,1-10。(B)载体体外转录本:第一口井包含1 kbp标记,后跟载体转录1-10。在装车前,在95°C下加热5分钟,使载波转录。<a href="https://www.jove.com/files/ftp_upload/59805/59805fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59805/59805fig3.jpg"> 图3:代表性DNA质量(高灵敏度DNA芯片)的SLIC-CAGE在第一轮载体降解前的痕迹。<a href="https://www.jove.com/files/ftp_upload/59805/59805fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59805/59805fig4.jpg"> 图4:PCR扩增后的SLIC-CAGE库的代表性DNA质量(高灵敏度DNA芯片)痕迹。(A) SLIC-CAGE 库需要额外的大小选择来删除短片段。(B) SLIC-CAGE 库在尺寸选择后使用 0.6x SPRI 磁珠与采样比。(C) 输出量较低的 SLIC-CAGE 库,需要选择大小以去除短片段。(D) SLIC-CAGE 库在选择尺寸后输出量较低,使用 0.6:1 SPRI 磁珠与采样比。<a href="https://www.jove.com/files/ftp_upload/59805/59805fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59805/59805fig5.jpg"> 图 5:SLIC-CAGE 库的验证。(A)在 SLIC-CAGE 库中标记簇间宽度的分布,这些宽度从S.cerevisae总 RNA 的 1、5 或 10 ng 制备,并在 nAnT-iCAGE 库中从 5 μg 的S. cerevisae总 RNA 制备。1 ng SLIC-CAGE 库中的大量窄标记群集表示其低复杂性。(B) 在S. s. SLIC-CAGE 库中用于 CTSS 标识的 ROC 曲线。所有S. cerevisae nAnT-iCAGE CTSS 都用作真正的集。(C) 在S. cerevisae纳米库中用于 CTSS 识别的 ROC 曲线.所有S. cerevisae nAnT-iCAGE CTSS 都用作真正的集。ROC曲线的比较表明,SLIC-CAGE在CTSS识别中优于纳米凯奇。使用了阵列快递E-MTAB-6519的数据。<a href="https://www.jove.com/files/ftp_upload/59805/59805fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
表1:载体合成基因序列。I-SceI 网站为粗体,斜体为紫色,I-CeuI 识别站点为绿色。<a href="https://www.jove.com/files/ftp_upload/59805/Table1.xlsx">请点击此处下载此文件。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>载体</td>
			<td colspan="2">反向引漆 5'-3'</td>
			<td>PCR 产品长度 / bp</td>
		</tr>
<tr>
<td>1</td>
			<td colspan="2">PCR_N6_r1: NNNNCTACGTCGCGAATT</td>
			<td>1034</td>
		</tr>
<tr>
<td>2</td>
			<td colspan="2">PCR_N6_r2: NNNNATATATATATATATATATGGGGGGGGGGGG</td>
			<td>966</td>
		</tr>
<tr>
<td>3</td>
			<td colspan="2">PCR_N6_r3: NNNNCACTGGGATCTCTTCG</td>
			<td>889</td>
		</tr>
<tr>
<td>4</td>
			<td colspan="2">PCR_N6_r4: NNNNNGCCGTCGATATATTTTCGT</td>
			<td>821</td>
		</tr>
<tr>
<td>5</td>
			<td colspan="2">PCR_N6_r5: NNNNNNNATGCAAGTCTTC</td>
			<td>744</td>
		</tr>
<tr>
<td>6</td>
			<td colspan="2">PCR_N6_r6: NNNNNGTGAATATCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT</td>
			<td>676</td>
		</tr>
<tr>
<td>7</td>
			<td colspan="2">PCR_N6_r7: NNNNNCGCGCTCCCTCATAC</td>
			<td>599</td>
		</tr>
<tr>
<td>8</td>
			<td colspan="2">PCR_N6_r8: NNNNNNTATACGTTTTCGTAC</td>
			<td>531</td>
		</tr>
<tr>
<td>9</td>
			<td colspan="2">PCR_N6_r9: NNNNNACCGCCGCCCCCGCGG</td>
			<td>454</td>
		</tr>
<tr>
<td>10</td>
			<td colspan="2">PCR_N6_r10: NNNNNCAGGACATTTTGCCCCACAA</td>
			<td>386</td>
		</tr>
<tr>
<td></td>
			<td colspan="2">• 所有载体模板的正向底漆都相同。下划线是 T7 启动子序列。PCR_GN5_f1: 塔塔塔CTCTCTA</td>
			<td></td>
		</tr>
</tbody></table>表2:载体模板放大的引信。对于所有载波模板,正向底漆都相同。下划线是 T7 启动子序列。PCR_GN5_f1: TAATACGACTCACTATAGNNNNCAGCGTTCTA.使用不同的反向引漆,产生PCR模板,从而产生不同长度的载波RNA。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>载体</td>
			<td>长度</td>
			<td>未封顶/μg</td>
			<td>盖/μg</td>
		</tr>
<tr>
<td>1</td>
			<td>1034</td>
			<td>3.96</td>
			<td>0.45</td>
		</tr>
<tr>
<td>2</td>
			<td>966</td>
			<td>8.36</td>
			<td>0.95</td>
		</tr>
<tr>
<td>3</td>
			<td>889</td>
			<td>4.4</td>
			<td>0.5</td>
		</tr>
<tr>
<td>4</td>
			<td>821</td>
			<td>6.6</td>
			<td>0.75</td>
		</tr>
<tr>
<td>5</td>
			<td>744</td>
			<td>4.4</td>
			<td>0.5</td>
		</tr>
<tr>
<td>6</td>
			<td>676</td>
			<td>3.08</td>
			<td>0.35</td>
		</tr>
<tr>
<td>7</td>
			<td>599</td>
			<td>4.4</td>
			<td>0.5</td>
		</tr>
<tr>
<td>8</td>
			<td>531</td>
			<td>3.96</td>
			<td>0.45</td>
		</tr>
<tr>
<td>9</td>
			<td>454</td>
			<td>2.64</td>
			<td>0.3</td>
		</tr>
<tr>
<td>10</td>
			<td>386</td>
			<td>2.2</td>
			<td>0.25</td>
		</tr>
</tbody></table>表3:RNA载体混合物。载波混合物共 49 μg 0.3-1 kbp:未封顶 = 44 μg,上限 = 5 μg。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>总RNA输入/ng</td>
			<td>PCR 周期</td>
		</tr>
<tr>
<td>1 纳克</td>
			<td>18</td>
		</tr>
<tr>
<td>2 纳克</td>
			<td>17</td>
		</tr>
<tr>
<td>5 纳克</td>
			<td>16</td>
		</tr>
<tr>
<td>10 纳克</td>
			<td>15-16</td>
		</tr>
<tr>
<td>25 纳克</td>
			<td>14-15</td>
		</tr>
<tr>
<td>50 纳克</td>
			<td>13-15</td>
		</tr>
<tr>
<td>100 纳克</td>
			<td>12-14</td>
		</tr>
</tbody></table>表 4:样品总RNA输入依赖性中PCR周期的预期数量。近似周期数基于使用糖精菌、果蝇黑色素和肌肉总RNA进行的实验。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>总RNA输入/纳克</td>
			<td>总体映射百分比</td>
			<td>% 唯一映射</td>
			<td>百分比载波</td>
		</tr>
<tr>
<td>1 纳克</td>
			<td>30</td>
			<td>20-30</td>
			<td>30</td>
		</tr>
<tr>
<td>2 纳克</td>
			<td>60</td>
			<td>20-50</td>
			<td>10</td>
		</tr>
<tr>
<td>5 纳克</td>
			<td>60-70</td>
			<td>40-60</td>
			<td>5-10</td>
		</tr>
<tr>
<td>10 纳克</td>
			<td>60-70</td>
			<td>40-60</td>
			<td>5-10</td>
		</tr>
<tr>
<td>25 纳克</td>
			<td>65-80</td>
			<td>40-70</td>
			<td>0-5</td>
		</tr>
<tr>
<td>50 纳克</td>
			<td>65-80</td>
			<td>40-70</td>
			<td>0-3</td>
		</tr>
<tr>
<td>100 纳克</td>
			<td>70-85</td>
			<td>40-70</td>
			<td>0-2</td>
		</tr>
</tbody></table>表5:预期映射效率和RNA总输入量的依赖程度。根据使用糖精和肌肉总RNA进行的实验,给出了近似数字。

Watch this Scientific Journal Video about Transcription Start Site Mapping Using Super-low Input Carrier-CAGE at JoVE.com

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

基因表达(CAGE)帽分析是一种对mRNA 5'ends进行全基因组定量映射的方法,以单核苷酸分辨率捕获RNA聚合酶II转录起始位点。本作品描述了一种低输入(SLIC-CAGE)协议,用于使用纳米图总RNA生成高质量库。

使用超低输入载波保持架进行转录启动站点映射

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). ...

这种方法可以帮助回答基因调控领域的关键问题，因为它使核心启动剂和增强剂发现使用少量的起始材料。该技术的主要优点是，它允许RNA聚合酶II转录启动位点使用10纳米的总RNA，无偏、全基因组的单核苷酸分辨率图谱。CAGE 被证明是无价的发现疾病相关的转录开始点，可以用作诊断标记。SLIC-CAGE 将这些发现扩展到扩大样本（如组织活检）。SLIC-CAGE 非常适合对脆弱细胞类型进行深入和高分辨率的启动子分析，包括早期胚胎发育阶段或来自各种模型生物体的胚胎组织。由于此协议中有许多步骤，因此在管间传输时注意检索尽可能多的材料，从而最大限度地减少每个步骤中的样品损失至关重要。首先准备每个模板的 PCR 组合。结合缓冲液，dNTP，独特的前底，模板质粒与合成载体基因，和融合聚合体根据手稿方向。通过移液混合试剂。将 PCR 混合物的 90 微升添加到每个反向底头和混合的 10 微升中。有关热循环条件，请参阅手稿。对感兴趣的RNA进行逆转录或RT。将一微升逆转录底转、10纳米RNA和4，990纳米的载体混合组合在10微升的总体积中。通过上下移液混合。将混合物加热到65摄氏度5分钟，然后立即放在冰上。根据稿稿说明准备RT混合物，并将混合的28微升加入10微升RNA。通过移液混合管内，直到均匀。在热循环器中运行 RT。一旦逆转录完成，用DNase和无RNase的SPRI磁珠净化产品。以 1.8 比 1 的体积比将珠子添加到 RT 混合物中，然后通过移液混合。让混合物在室温下坐五分钟。然后，将珠子放在磁性支架上，让它们分开五分钟。丢弃上一杯，在珠子中加入200微升新鲜准备好的70%乙醇。不要混合珠子或将其从磁性支架上取下，并在添加后立即取出乙醇。将管子放在磁性支架上，用 P10 移液器将任何剩余的液滴推出，从而去除所有乙醇痕迹。立即加入42微升水，并上下移液器60次，以利特样品，注意不要引起发泡。在37摄氏度下孵育管，不带盖子5分钟，然后分离磁性支架上的珠子。然后，将上流水液转移到一组新的管子中，注意取回所有上流液，但避免珠子结转。RNase I治疗后，将45微升样品与105微升的制备的链球菌珠混合，在37摄氏度下孵育30分钟。在孵育过程中，每10分钟移液一次样品混合。然后，将磁支架上的磁珠分离并取出上经剂。用缓冲器 A、B 和 C 清洗珠子，从缓冲器 A 开始，将珠子重新在 150 微升缓冲器中重新填充。在磁性支架上分离两到三分钟，然后取出上经剂。将缓冲液 B 和 C 预热至 37 摄氏度，然后重复洗涤步骤。要去除所有非封盖RNA，必须彻底清洗链球菌珠和管壁，并在添加下一个之前完全去除洗涤缓冲液。要释放cDNA，将珠子重新在35微升1X RNase I缓冲和孵育，在95摄氏度下孵育5分钟。孵育后，立即将珠子放在冰上两分钟。将磁支架上的珠子分离，然后将上经器转移到一组新的管子上。将珠子重新悬浮在 30 微升 1X RNase I 缓冲器中，然后在磁性支架上分离。将上一液与以前收集的上一级机结合，总体积约为 65 微升。执行 qPCR 以确定目标库放大的 PCR 周期数。准备 qPCR 主混合，以放大来自载体的整个 DNA 库。根据手稿方向设置热循环条件，并放大制备的样品。SLIC-CAGE 协议使得从起始RNA材料的纳米图获得测序就绪库成为可能。最终库中的片段长度介于 200 到 2，000 个基对之间。较短的片段是 PCR 伪影，并可能导致排序问题。可以执行其他大小选择轮以将其删除。与 NanoCAGE 相比，SLIC-CAGE 在转录启动位识别方面表现出显著更好的性能，这从两种技术对不同数量的 S.cerevisiae 总RNA进行的接收器操作特性曲线中可见一些。在执行此协议时，必须记住，示例丢失可能会导致低复杂性库。为了防止这种情况，必须使用低结合移液器尖端和管子。如果没有SLIC-CAGE，到目前为止，各种细胞类型的启动子分析一直无法访问。这包括分析胚胎发育阶段或胚胎组织从广泛的模型生物体。

Research

JoVE Journal

Genetics

In Vitro Transcription Assays and Their Application in Drug Discovery

In Vitro Transcription Assays and Their Application in Drug Discovery

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

使用BrUTP链特异性转录运行的（TRO）方法终止转录分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

逆转录病毒扫描：映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

使用基于循环 RT-PCR 的策略对玉米 Mitochondrion 中初级和已处理转录的区分和映射

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...