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	<li>Addepalli, B., Venus, S., Thakur, P., Limbach, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+ribonuclease+activity+of+cusativin+from+Cucumis+sativus+for+mapping+nucleoside+modifications+in+RNA.">Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.</a> Analytical and Bioanalytical Chemistry. 409 (24), 5645-5654 (2017).</li><li>Gao, H., Liu, Y., Rumley, M., Yuan, H., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+confirmation+of+chemically+modified+RNAs+using+exonuclease+digestion+and+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry.">Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 23 (21), 3423-3430 (2009).</li><li>McLuckey, S. A., Van Berkel, G. J., Glish, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+mass+spectrometry+of+small,+multiply+charged+oligonucleotides.">Tandem mass spectrometry of small, multiply charged oligonucleotides.</a> Journal of The American Society for Mass Spectrometry. 3 (1), 60-70 (1992).</li><li>Fountain, K. J., Gilar, M., Gebler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+native+and+chemically+modified+oligonucleotides+by+tandem+ion-pair+reversed-phase+high-performance+liquid+chromatography/electrospray+ionization+mass+spectrometry.">Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 17 (7), 646-653 (2003).</li><li>Taucher, M., Breuker, K. <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+modified+RNA+by+top-down+mass+spectrometry.">Characterization of modified RNA by top-down mass spectrometry.</a> Angewandte Chemie International Edition in English. 51 (45), 11289-11292 (2012).</li><li>Kellner, S., Burhenne, J., Helm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+RNA+modifications.">Detection of RNA modifications.</a> RNA Biology. 7 (2), 237-247 (2010).</li><li>Thomas, B., Akoulitchev, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+of+RNA.">Mass spectrometry of RNA.</a> Trends in Biochemical Sciences. 31 (3), 173-181 (2006).</li><li>Bjorkbom, 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=Bidirectional+direct+sequencing+of+noncanonical+RNA+by+two-dimensional+analysis+of+mass+chromatograms.">Bidirectional direct sequencing of noncanonical RNA by two-dimensional analysis of mass chromatograms.</a> Journal of the American Chemical Society. 137 (45), 14430-14438 (2015).</li><li>Zhang, 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=A+general+LC-MS-based+RNA+sequencing+method+for+direct+analysis+of+multiple-base+modifications+in+RNA+mixtures.">A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.</a> Nucleic Acids Research. 47 (20), 125 (2019).</li><li>Zhang, 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=Direct+sequencing+of+tRNA+by+2D-HELS-AA+MS+Seq+reveals+its+different+isoforms+and+dynamic+base+modifications.">Direct sequencing of tRNA by 2D-HELS-AA MS Seq reveals its different isoforms and dynamic base modifications.</a> ACS Chemical Biology. 15 (6), 1464-1472 (2020).</li><li>Bakin, A., Ofengand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+newly+located+pseudouridylate+residues+in+Escherichia+coli+23S+ribosomal+RNA+are+all+at+the+peptidyltransferase+center:+analysis+by+the+application+of+a+new+sequencing+technique.">Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique.</a> Biochemistry. 32 (37), 9754-9762 (1993).</li><li>Cantara, W. 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=The+RNA+Modification+Database,+RNAMDB:+2011+update.">The RNA Modification Database, RNAMDB: 2011 update.</a> Nucleic Acids Research. 39 (Database issue), D195-D201 (2011).</li></ol>

<table><tbody><tr><td>5&#039; DNA Adenylation kit</td><td>New England Biolabs</td><td>E2610S</td><td>50uM concentration</td></tr><tr><td>6550 Q-TOF mass spectrometer</td><td>Agilent Technologies</td><td>5991-2116EN</td><td>Coupled to a 1290 Infinity LC system</td></tr><tr><td>A(5&amp;acute;)pp(5&amp;acute;)Cp-TEG-biotin-3&amp;acute;</td><td>ChemGenes</td><td>91718</td><td>HPLC purified</td></tr><tr><td>ATP&amp;gamma;S</td><td>Sigma-Aldrich</td><td>11162306001</td><td>Lithium salt</td></tr><tr><td>Bicine</td><td>Sigma-Aldrich</td><td>B8660</td><td>BioXtra, &amp;ge;99% (titration)</td></tr><tr><td>Biotin maleimide</td><td>Vector Laboratories</td><td>SP-1501</td><td>Long arm</td></tr><tr><td>C18 column</td><td>Waters</td><td>186003532</td><td>50 mm &amp;times; 2.1 mm Xbridge C18 column with a particle size of 1.7 &amp;mu;m</td></tr><tr><td>Centrifugal Vacuum Concentrator</td><td>Labconco</td><td>Refrig 115v/60hz 7310022</td><td>Labconco CentriVap</td></tr><tr><td>ChemBioDraw</td><td>PerkinElmer</td><td>ChemDraw Prime</td><td>Generate a chemical structure and property data of structures &amp;amp; fragments</td></tr><tr><td>CMC (N-cyclohexyl-N?-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate)</td><td>Sigma-Aldrich</td><td>2491-17-0</td><td>95% Purifiy</td></tr><tr><td>Cyanine3 maleimide (Cy3)</td><td>Lumiprobe</td><td>11080</td><td>Water insoluble</td></tr><tr><td>DEPC-treated water</td><td>Thermo Fisher Scientific</td><td>AM9906</td><td>Autoclaved, certified nuclease-free</td></tr><tr><td>Diisopropylamine (DIPA)</td><td>Thermo Fisher Scientific</td><td>108-18-9</td><td>99% Alfa Aesar</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855</td><td>Anhydrous dimethyl sulfoxide, 99.9%</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td>Anhydrous, crystalline, BioReagent, suitable for cell culture</td></tr><tr><td>Formic acid</td><td>Merck</td><td>64-18-6</td><td>98-100%, ACS reag, Ph Eur</td></tr><tr><td>Hexafluoro-2-propanol (HFIP)</td><td>Thermo Fisher Scientific</td><td>920-66-1</td><td>99% Acros Organics</td></tr><tr><td>LC-MS sample vials</td><td>Thermo Fisher Scientific</td><td>C4000-11</td><td>Plastic screw thread vials</td></tr><tr><td>LC-MS vial caps</td><td>Thermo Fisher Scientific</td><td>C5000-54A</td><td>Autosampler vial screw thread caps</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt; buffer</td><td>Sigma-Aldrich</td><td>88975</td><td>BioUltra, &gt;0.1 M Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;, &gt;0.2 M NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Oligo Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4060</td><td>Spin column</td></tr><tr><td>OriginLab</td><td>OriginLab</td><td>OriginPro</td><td>Data analysis and graphing software</td></tr><tr><td>pCp-biotin</td><td>TriLink BioTechnologies</td><td>NU-1706-BIO</td><td>20 ul (1 mM)</td></tr><tr><td>RNA #1--#6</td><td>Integrated DNA Technologies</td><td>Custom RNA oligos</td><td>19nt-21nt single-stranded RNAs, used without further purification</td></tr><tr><td>Rocking platform shaker</td><td>VWR</td><td>Orbital Shaker Standard 1000</td><td>Speed Range 40 to 300 rpm</td></tr><tr><td>Streptavidin magnetic beads</td><td>Thermo Fisher Scientific</td><td>88816</td><td>Binding approx. 55ug biotinylated rabbit lgG per mg of beads</td></tr><tr><td>Sulfonated Cyanine3 maleimide</td><td>Lumiprobe</td><td>11380</td><td>Water soluble</td></tr><tr><td>T4 DNA ligase 1</td><td>New England Biolabs</td><td>M0202S</td><td>400 units/uL</td></tr><tr><td>T4 polynucleotide kinase</td><td>Sigma-Aldrich</td><td>T4PNK-RO</td><td>From phage T4 am N81 pse T1 infected Escherichia coli BB</td></tr><tr><td>Tris-HCl buffer</td><td>Sigma-Aldrich</td><td>T6455</td><td>Tris-HCl Buffer, pH 10, 10&amp;times;, Antigen Retriever</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>81871</td><td>Urea for synthesis. CAS No. 57-13-6, EC Number 200-315-5.</td></tr></tbody></table>

2d-hels ms seq: a general lc-ms-based method for direct and de novo sequencing of rna mixtures with different nucleotide modifications

Watch this Scientific Journal Video about 2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications at JoVE.com

<meta charset="utf8"></head><body>2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

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

2d-hels-ms-seq-general-lc-ms-based-method-for-direct-de-novo

Research

JoVE Journal

Genetics

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

1.8K 조회수.  New York Institute of Technology. 여기에서는 cDNA 중간체 없이 짧은 RNA(실행당 

비디오: 2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

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

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

질량 분석 기반 식별 RNA 바인딩 영역에 대 한 샘플 준비

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

연구

생화학

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.

작은 RNA-seq 개선: 2'-O-메틸 RNA의 더 적은 편견 및 더 나은 검출

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 ...

유전학

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

그람 양성 박테리아에서 RNA 염기서열분석과 결합된 MS2-선호도 정화

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

AQRNA-seq를 사용한 tRNA 역학 분석

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

저자 스포트라이트: AQRNA-seq의 Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms (작은 RNA 매핑 및 단백질 번역 메커니즘 규명에서 AQRNA-seq 역할)

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

small RNA 정량을 위한 AQRNA-seq

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

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.

RNA의 1 H R 1θ 이완 분산 실험의 샘플 준비 및 설치의 실제적인 양상

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

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

디메틸 설페이트를 사용한 RNA 구조 프로빙 시험관 내 및 세포에서 시퀀싱을 사용한 돌연변이 프로파일링

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

10 나노 그램 총 RNA의 단일 읽기와 결합하여 최종 mRNA - Seq의 Illumina 도서관

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

생물학

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.

RNA 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로

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

2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

질량 분석 기반 식별 RNA 바인딩 영역에 대 한 샘플 준비

작은 RNA-seq 개선: 2'-O-메틸 RNA의 더 적은 편견 및 더 나은 검출

그람 양성 박테리아에서 RNA 염기서열분석과 결합된 MS2-선호도 정화

small RNA 정량을 위한 AQRNA-seq

small RNA 정량을 위한 AQRNA-seq

small RNA 정량을 위한 AQRNA-seq

RNA의 1 H R _1θ 이완 분산 실험의 샘플 준비 및 설치의 실제적인 양상

디메틸 설페이트를 사용한 RNA 구조 프로빙 시험관 내 및 세포에서 시퀀싱을 사용한 돌연변이 프로파일링

10 나노 그램 총 RNA의 단일 읽기와 결합하여 최종 mRNA - Seq의 Illumina 도서관

RNA 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로