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

Summary

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.

Abstract

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 organism^1,2 . 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.

Protocol

A flowchart outlining this protocol is displayed in Figure 1.

1. Treatment of Cells with Thrombin, RNA Isolation, Quality Assessment and Quantification of RNA

1. 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).
2. Change media to the starvation media (0% FBS) 30 min prior to treatment with thrombin.
3. Treat the cells with 0.05 U/ml thrombin or leave untreated as a control for 6 hr at 37 °C and 5% CO₂.
4. Isolate total RNA from the treated and contr....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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