Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Summary

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Abstract

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N₂ gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O₂ and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O₂-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Introduction

Many organisms respond to hypoxia, or low O₂, by altering gene expression ^1,2,3. This response helps cells cope with the lack of a substrate critical for aerobic respiration and for several biosynthetic reactions, but also with a changing redox state ⁴. Several microarray studies performed in S. cerevisiae show that the mRNA levels of hundreds of genes change in response to hypoxia ^5,6,7,8,

Protocol

1. Inducing Hypoxia

1. One day or more before the hypoxia time course: Prepare the incubator, cell filtering system, vacuum, gas tank, flasks, stoppers, glass tubing, and tubing, as in the Materials Table.
2. Place the N₂ tank, incubator, vacuum and filtering system in close proximity, to enable quick processing of cells.
3. Prepare sterile liquid YPD media (1% Yeast Extract, 2% peptone, 2% glucose) by mixing the components in a glass bottle and autoclaving.
4. Plan layout of flasks in the incubator.
   NOTE: From the N₂ tank, the first flask will be a water trap, the second flask will be the las....

Results

The hypoxia time course and RNA-seq analysis were performed independently three times. To examine the reproducibility of the three replicates, gene expression data for all genes was analyzed using Principal Component Analysis (PCA). Figure 2 shows how the samples change over the first two principal components, which together represent 58.9% of the variability. This analysis indicated that each time course exhibits similar changes (as depicted by the similar s.......

Discussion

In this study, the mRNA levels for all genes was measured during hypoxia in the yeast S. cerevisiae. The goal was to analyze how global gene expression changes due to growth in a controlled near-anoxic environment. Several steps were taken to ensure that the method described here was carefully controlled and reproducible. First, cells were exposed to a precisely defined hypoxic environment: 99.999% N₂ in rich media (YPD). Other studies of hypoxia have closed off the flask or tube to air

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Enclosed dry incubator	Thermo Scientific	MaxQ 4000	Set at 30 °C. Modify the door to allow entry of one Tygon tube. Alternatively, use the New Brunswick G25 incubator, which contains a tube port. Do not use an open-air water shaker, as condensation will collect in the tubes between flasks, possibly cross-contaminating cultures.
Micro-analysis Filter Holder	Millipore	XX1002530	25mm diameter, stainless steel support, no. 5 perforated silicone stopper mounts in standard 125 mL filtering flask
Strong vacuum	Edwards	E-LAB 2	The “house” vacuum may be too weak. Alternatively, use an electric-power portable vacuum pump like the one listed here.
1,000 mL flask			To act as vacuum trap.
~2 foot lengths of Heavy Wall Vacuum Tubing, inner diameter 3/8 in, outer diameter 7/8 in	Tygon	38TT	Two pieces: the first connects vacuum to trap, and the second connects trap to filter system.
High-pressure N2 gas tank			99.999% purity, >1,000 psi, with a regulator and gas flow controller
Autoclaved 500 mL flask			Opening covered with aluminum foil. One for each yeast strain.
Autoclaved 250 mL flasks			Openings covered with aluminum foil. One for each time point plus two for water traps.
Flask stoppers (size 6, two holes with 5 mm diameter)			Sterilized with 70% ethanol. One for each flask.
Glass tubing, length 9 cm or 17 cm, inner diameter 2 mm, outer diameter 5 mm			Sterilized with 70% ethanol. Two tubes for each flask. Place into the holes of each stopper. See Figure 1 for placement of 9- vs 17- cm tubes.
~25 cm lengths of plastic tubing, inner diameter 5 mm	Tygon	E-3603	One piece for each flask. Sterilized with 70% ethanol.
Sterile filter discs	Millipore	HAWP02500	25 mm diameter, 0.45 µm pore size, one for each time point
Sterile dH2O (~100 mL)
1 mL cuvettes			For measuring OD600 (i.e., cell concentration)
50 mL sterile centrifuge tubes			One for each time point
Clean and sterile tweezers
liquid nitrogen			For freezing cells
acid-washed beads	Sigma	G8772	Keep at 4 °C for lysing cells
Qiagen RNeasy Mini Kit	Qiagen	74104	For RNA column purification
Qiagen RLT buffer			Prepare by adding 10 µL of β-Mercaptoethanol per 1 mL of RLT buffer, keep at 4 °C.
2 mL collection tubes	Qiagen		included in the Qiagen Rneasy Mini Kit
Buffer RPE	Qiagen		included in the Qiagen Rneasy Mini Kit
Buffer RW1	Qiagen		included in the Qiagen Rneasy Mini Kit
DNase I stock and working solutions	Qiagen	79254	The DNase I enzyme comes as lyophilized powder in a glass vial. Using a sterile needle and syringe, inject 550 µL of RNase-free water (provided in Qiagen kit) into the vial. Mix by gently inverting the bottle. To avoid denaturing the enzyme, do not vortex. Using a pipet, remove this stock solution from the vial and store in freezer (-20 °C) in single-use aliquots (80 µL each). The stock solution should not be thawed and refrozen.
Buffer RDD	Qiagen		included in the Qiagen DNase Kit
Ice cold 2 mL screw-cap tubes			For lysing cells during RNA extraction
bead mill homogenizer	Biospec Mini-Beadbeater-24	112011	Keep in cold room
Bacto Peptone	BD	DF0118	for liquid YPD media
Bacto Yeast Extract	BD	DF0886	for liquid YPD media
glucose	Fisher	D16	for liquid YPD media
Qubit assay tubes	Thermo Fisher	Q32856	for measuring nucleic acid concentration
Quant-iTTM dsDNA BR Assay Kit	Thermo Fisher	Q32853	for measuring nucleic acid concentration
Quant-iTTM RNA Assay Kit	Thermo Fisher	Q32855	for measuring nucleic acid concentration
Qubit Fluorometer	Thermo Fisher	Q33216	for measuring nucleic acid concentration
Commercial electrophoresis system	Agilent	Bioanalyzer 2100	for measuring nucleic acid quality
Next-generation sequencer	Illumina	HiSeq 2500	for sequencing libraries
automated liquid handling system	Wafergen	Apollo 324	for creating sequencing libraries
PrepX PolyA mRNA Isolation Kit	Wafergen	400047	for isolating mRNA from total RNA
PrepX RNA SEQ for Illumina Library Kit	Wafergen	400039	for creating strand-specific sequencing libraries from total RNA
Barcode Splitter			https://toolshed.g2.bx.psu.edu/repository?repository_id=7119c4f7a89efa57&changeset_revision=e7b7cdc1834d
Samtools, which includes the gzip command			http://www.htslib.org/download/
Trimmomatic			http://www.usadellab.org/cms/?page=trimmomatic
Bowtie2 (installed before TopHat)			http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
TopHat			https://ccb.jhu.edu/software/tophat/index.shtml
HTSeq			http://www-huber.embl.de/HTSeq/doc/overview.html
R (installed before R Studio)			https://cran.rstudio.com
R Studio (free version)			https://www.rstudio.com/products/rstudio/download/