Name: rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells
Uploaded: 2013-02-13T13:00:00
Description: Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

The authors would like to thank Dr. Stephen Kingsmore and the Pediatric Genome Medicine Center at Children's Mercy Hospitals and Clinics for the use of their computing clusters for our data analysis, Illumina's field service team (Elizabeth Boyer, Scott Cook and Mark Cook) and technical consultant team for their quick responses and helpful suggestions on the running of the next generation DNA sequencing instrument, HiScanSQ, and data quality analysis. This work was supported in part by National Institutes of Health Grant HL080042 (to S.Q.Y.) and start-up fund and endowment of Children's Mercy Hospitals and Clinics, University of Missouri at Kansas City (to S.Q.Y.).

A flowchart outlining this protocol is displayed in Figure 1.
1. Treatment of Cells with Thrombin, RNA Isolation, Quality Assessment and Quantification of RNA
<ol>
	<li>Culture Human Lung Microvascular Endothelial Cells (HMVEC-LBl) to 90-100% confluence in 6-well plates in EGM-2 medium with 5% FBS, growth factors and antibiotics (Lonza, cat# CC-3202).</li>
	<li>Change media to the starvation media (0% FBS) 30 min prior to treatment with thrombin.</li>
	<li>Treat the cells with ...

<ol>
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Key steps
RNA Handling: RNases will degrade even the most high-quality RNA, therefore care must be taken during the isolation, storage and use of RNA10. Gloves are always worn to prevent contamination by RNases found on human hands. Gloves should be changed often, particularly after touching skin, doorknobs or other common surfaces. A set of pipettes should be dedicated solely to RNA work and all tips and tubes should be RNase-free. RNA isolation and downstream applica...

<table border="1">
 <tbody>
 <tr>
 <td width="183">Reagents or Equipments</td>
 <td width="85">Company</td>
 <td width="185">Catalog number</td>
 <td width="359">Comments</td>
 </tr>
 <tr>
 <td>Human Lung Microvascular Endothelial Cells</td>
 <td>Lonza</td>
 <td>CC-2815</td>
 <td></td>
 </tr>
 <tr>
 <td>Lonza, Bullet Kit</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td>Contains EGM-2, FBS, growth factors and antibiotics</td>
 </tr>
 <tr>
 <td>Thrombin</td>
 <td>Sigma</td>
 <td>T4393</td>
 <td></td>
 </tr>
 <tr>
 <td>Ambion mirVana Kit</td>
 <td>Life Technologies</td>
 <td>AM 1560</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase-Zap</td>
 <td>Life Technologies</td>
 <td>AM9782</td>
 <td></td>
 </tr>
 <tr>
 <td>Experion StdSens RNA</td>
 <td>Bio-Rad</td>
 <td>700-7103</td>
 <td></td>
 </tr>
 <tr>
 <td>TruSeq RNA Preparation Kit</td>
 <td>Illumina</td>
 <td>FC-122-1001</td>
 <td></td>
 </tr>
 <tr>
 <td>AMPureXP Beads</td>
 <td>Beckman Coulter</td>
 <td>A63881</td>
 <td></td>
 </tr>
 <tr>
 <td>Superscript Reverse Transcriptase II</td>
 <td>Life Technologies</td>
 <td>18064-014</td>
 <td></td>
 </tr>
 <tr>
 <td>Experion DNA 1K</td>
 <td>Bio-Rad</td>
 <td>700-7107</td>
 <td></td>
 </tr>
 <tr>
 <td>QuantiTect SyberGreen</td>
 <td>Qiagen</td>
 <td>204163</td>
 <td></td>
 </tr>
 <tr>
 <td>PE Cluster Generation Kit</td>
 <td>Illumina</td>
 <td>PE-401-3001</td>
 <td></td>
 </tr>
 <tr>
 <td>PhiX Control Kit</td>
 <td>Illumina</td>
 <td>FC110-301</td>
 <td></td>
 </tr>
 <tr>
 <td>200 Cycle SBS Kit</td>
 <td>Illumina</td>
 <td>FC-401-3001</td>
 <td></td>
 </tr>
 <tr>
 <td>HiScanSQ* </td>
 <td>Illumina </td>
 <td>SY-103-2001 </td>
 <td></td>
 </tr>
 <tr>
 <td>cBot</td>
 <td>Illumina</td>
 <td>SY-301-2002</td>
 <td></td>
 </tr>
 <tr>
 <td>qPCR machine - Viia7</td>
 <td>Life Technologies</td>
 <td>Model #VIIA7 / Equipment #10631261</td>
 <td>Or equivalent </td>
 </tr>
 <tr>
 <td>Experion System </td>
 <td>Bio Rad</td>
 <td>7007001</td>
 <td>Bioanalyzer is an alternative system</td>
 </tr>
 <tr>
 <td>Spectrophotometer </td>
 <td>Bio-Tek</td>
 <td>Epoch Microplate Spectrophotometer</td>
 <td>Or equivalent</td>
 </tr>
 <tr>
 <td>Centrifuge - Sorvall Legend XTR </td>
 <td>Thermo Scientific</td>
 <td>75004521</td>
 <td>Or equivalent</td>
 </tr>
 <tr>
 <td>Magnetic stand</td>
 <td>Life Technologies</td>
 <td>AM10027</td>
 <td></td>
 </tr>
 <tr>
 <td>96-well thermocycler</td>
 <td>General Lab Supplier</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 3. List of Key Reagents and Major Equipment. *, In the video, HiSeq1000 instead of HiScanSQ was demonstrated.</td>
 </tr>
 </tbody>
</table>

rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

For Step 1: The 28s:18s ratio is traditionally used as an indicator of RNA degradation. Ideally, the 28s peak should have approximately twice the area of the 18s band (a ratio of 2), however this ideal ratio is often not seen in practice. Furthermore, 28s:18s ratios obtained from spectrophotometric methods can underestimate the amount of degradation of the RNA. To more accurately quantify the degradation, and therefore the quality of the RNA sample, the Experion system calculates an RNA Quality I...

Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

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