We thank the Lewis-Sigler Institute for Integrative Genomics Sequencing Core Facility at Princeton University for technical advice and for RNA library preparation and sequencing. This work was supported by grants from Rowan University and NIH NIGMS R15GM113187 to M.J.H.

1. Inducing Hypoxia
<ol>
	<li>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.</li>
	<li>Place the N2 tank, incubator, vacuum and filtering system in close proximity, to enable quick processing of cells.</li>
	<li>Prepare sterile liquid YPD media (1% Yeast Extract, 2% peptone, 2% glucose) by mixing the components in a glass bottle and autoclaving.</li>
	<li>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 last time point, the third flask will be the second-to-last time point and so on until the last flask which is a final water trap. Figure 1 shows the order and connections between the flasks.</li>
	<li>The day before the time course, inoculate a yeast colony into a culture of 5 mL liquid YPD within a sterile test tube. Specifically, pick up a full colony from a plate using a sterile applicator stick. Place the stick into the liquid YPD and shake the stick until most of the cells are in suspension.</li>
	<li>Rotate the tubes at 30 &#176;C overnight (~16 h). 
	NOTE: After 16 h, wildtype cells will achieve saturation (~2 x 108 cells/mL), indicated by a very cloudy culture. Other strains may take longer to reach saturation and should be tested by measuring cell concentration with a spectrophotometer, as indicated in Step 1.9.4.</li>
	<li>On the day of the time course, after 16 h of incubation, dilute the saturated culture 1:50 by using a 5 mL pipet to place 4 mL of overnight culture into 196 mL of liquid YPD within a sterile 500 mL flask. Grow with shaking at ~200 rpm for 4 h at 30 &#176;C. 
	NOTE: After 4 h, a wildtype culture will reach a mid-log concentration of ~1-2 x 107 cells/mL.</li>
	<li>During the 4 h incubation, label the flasks (for hypoxia and water traps) and the 50 mL collection tubes. If the incubator in step 1.7 above is not used for the hypoxia time course, turn on the other incubator and set to 30 &#176;C.</li>
	<li>Just before subjecting cells to hypoxia:
	<ol>
		<li>Add ~50 mL water to two of the sterile 250 mL flasks which will serve as the water traps.</li>
		<li>Fill 1/3 of an ice bucket with liquid nitrogen and submerge a polystyrene foam rack to hold 50 mL centrifuge tubes.</li>
		<li>Set up the filter system for collecting cells. Add a sterile filter disc onto the bottom unit of the filtration system using sterile tweezers, being careful to only touch the edges of the filter disc. Place the filter top unit onto the filter bottom unit and secure with the clamps provided with the system. Ensure that the bottom and top units are aligned so that there is a tight seal and no leakage.</li>
		<li>Measure the cell concentration with a spectrophotometer. Dilute cells so that they can be measured in the linear range of the spectrophotometer &#8211; OD600 between ~0.2 - 0.6, depending on the spectrophotometer. 
		NOTE: One OD600 unit is ~3 x 107 cells/mL, depending on cell morphology and spectrophotometer. A concentration of ~1-2 x 107 cells/mL is expected, corresponding to OD600 of ~0.33 - 0.66.</li>
		<li>Quickly obtain the time 0 sample.
		<ol>
			<li>Connect the filter system to a strong (~10 mbar) vacuum via vacuum tubing and a 1,000 mL flask trap. Turn on the vacuum. Pour the culture (usually ~20 mL) into the top unit and wait for liquid to be pulled through the filter (~10 s, depending on the strength of the vacuum).</li>
			<li>Carefully remove the filter disc with clean tweezers and place into a 50 mL centrifuge tube. Immediately place the centrifuge tube into the rack submerged in liquid nitrogen. Importantly, do not pre-chill the tubes before inserting the filter as the tubes will explode.</li>
			<li>After &#62;30 s, place the tube into a -80 &#176;C freezer for later RNA extraction. After each filtration, clean and reassemble the filter system. Rinse the system by pulling water through for 10 s without a filter disc. Finally, wipe dry the system with a paper towel.</li>
		</ol>
		</li>
		<li>Dilute the 4 h culture into different flasks for the various time points, so that each flask reaches mid-log concentration (~1-2 x 107 cells/mL) at the indicated time point of hypoxia. 
		NOTE: Table 1 shows dilutions that were used for the wild-type S288C HAP1+ haploid strain. It is recommended to test each strain in these hypoxic culture conditions and adjust the dilutions accordingly.</li>
		<li>Once cells and media are added to flasks, replace the aluminum foil covering the flask opening with a stopper containing two glass tubes inserted.</li>
		<li>Place the flasks in the incubator in the layout determined earlier.</li>
		<li>Securely connect the tubing as shown in Figure 1.</li>
		<li>Check that all the stoppers are tightly pushed into the flask openings.</li>
	</ol>
	</li>
	<li>At time 0, open the regulator valve. Then set the flow meter to 3 L/min. 
	NOTE: Bubbling will be observed in both water flasks, indicating proper gas flow. If this is not the case, check that all stoppers are tight and tubing is properly attached.</li>
	<li>Close the incubator and set the shaking speed to ~200 rpm. Start a timer.</li>
	<li>Before each time point, set up the filter system as describe above in step 1.9.3.</li>
	<li>At each time point (e.g., 5 min, 10 min, etc.), have two people quickly process the cells as follows:
	<ol>
		<li>First person: remove the appropriate flask from the holder and take out the stopper (with glass tubing attached). 
		NOTE: This will not break the flow of N2 to any of the remaining culture flasks but will temporarily break the flow to the last water trap.</li>
		<li>Second person: Pipet 1 mL of culture and dispense into a cuvette. Reconnect the tubing from the culture flask that is now at the end of the line to the last water trap (One tube and one stopper will be removed in the process.). Then, measure cell concentration in the cuvette, as described above in step 1.9.4.</li>
		<li>First person: Pour the remainder of the culture into the vacuum filtration system, and then perform filtering and freezing as described above in step 1.9.5.</li>
	</ol>
	</li>
	<li>When finished with all the time points, turn off the N2 gas. First close the regulator and wait for the pressure to release, and then turn off the flow meter. Finally, disassemble the system and clean all materials with water and then 70% ethanol.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Prepare RLT buffer for RNA column purification, as described in the Materials Table.</li>
	<li>Prepare a working solution of DNase by adding 10 &#181;L of the DNase stock solution to 70 &#181;L of buffer RDD per sample (see Materials Table).</li>
	<li>Remove the 50 mL tubes containing filters and cells from the -80 &#176;C freezer and place on ice to thaw (~15 min). Perform the following steps with tubes on ice.</li>
	<li>Label 2 mL screw-cap tubes and place them on ice, one tube for each sample. Add ~0.6 mL of acid-washed beads to each tube, using a 1.5 mL microcentrifuge tube to scoop and measure the beads.</li>
	<li>Add 0.6 mL of cold RLT buffer to the 50 mL tubes.</li>
	<li>Pipet up and down to remove cells from filter and suspend into solution. Avoid letting the filter remain in the solution as the filter will soak up the solution. Remove all solution absorbed into the filter by using the pipet tip to squeeze the filter against the wall of the tube.</li>
	<li>Transfer all of the liquid from the 50 mL tube to the screw-cap tubes containing beads.</li>
	<li>Place the screw-cap tubes into a bead mill homogenizer and run for one min. 
	Immediately place the tubes on ice for three min. Again, place the tubes into the homogenizer and run for one min.</li>
	<li>Remove the tubes from the homogenizer and place on ice for 5 min. The beads will settle to the bottom of the tubes.</li>
	<li>Perform the remainder of the steps at room temperature.</li>
	<li>With a pipet, transfer only the lysate (~350 &#181;L), avoiding the beads, to a new 1.5 mL microcentrifuge tube.</li>
	<li>Microcentrifuge for 2 min at max speed and then transfer the supernatant to a new 1.5 mL microcentrifuge tube.</li>
	<li>Add 1 volume of 70% ethanol (made from 200 proof molecular biology-grade ethanol). Mix well by pipetting.</li>
	<li>Transfer the solution and any precipitate to an RNA column that has been placed inside a 2 mL collection tube.</li>
	<li>Centrifuge for 15 s at &#8805;12,000 x g and discard the flow-through (the liquid in the tube).</li>
	<li>Add 350 &#181;L of buffer RW1 to the column. Centrifuge for 15 s at &#8805;12,000 x g and discard the flow-through.</li>
	<li>Add 80 &#181;L DNase I incubation mix to the column membrane (do not get on sides of tube) and allow to sit for 15 min.</li>
	<li>Add 350 &#181;L of buffer RW1 to column. Microcentrifuge for 15 s at &#8805;12,000 x g and discard the flow-through.</li>
	<li>Add 500 &#181;L of buffer RPE to the column. Microcentrifuge for 15 s at &#8805;12,000 x g and discard flow-through.</li>
	<li>Add 500 &#181;L of buffer RPE to the column. Microcentrifuge for 2 min at &#8805;12,000 x g.</li>
	<li>Carefully remove the column and place into a new 2 mL collection tube. Microcentrifuge at full speed for 1 min (to completely dry the membrane).</li>
	<li>Discard the collection tube and place the column into a 1.5 mL microcentrifuge tube. Add 30 &#181;L of RNase-free water to the column membrane.</li>
	<li>Elute the RNA from the column by microcentrifuging for 1 min at &#8805;12,000 x g. When loading the columns into the microcentrifuge, make sure the lids of the collection tube are facing in the direction the centrifuge spins, to prevent the lids breaking off.</li>
	<li>Add another 30 &#181;L of RNase-free water to the column membrane, while keeping the column in the same microcentrifuge tube.</li>
	<li>Microcentrifuge for 1 min at &#8805;12,000 x g, so that the final eluate volume is 60 &#181;L.</li>
	<li>Store each 1.5 mL tube containing 60 &#181;L of RNA in the -80 &#176;C freezer.</li>
</ol>
3. Determining RNA Concentration and Quality
<ol>
	<li>Measure the concentration of RNA and DNA in each RNA sample, using (a) a fluorometer, (b) DNA- and RNA-specific fluorescent dyes and (c) assay tubes, as listed in the Materials Table. Follow the instructions of the fluorometer and the dyes.</li>
	<li>Alternatively, measure the nucleic acid concentration with a UV spectrophotometer set at 260 nm. 
	NOTE: An A260 reading of 1.0 is equivalent to ~40 &#181;g/mL single-stranded RNA.</li>
	<li>Test RNA quality by running the RNA on a commercial nucleic acid analyzer (see Materials Table) or on a standard formaldehyde/agarose gel 26.</li>
</ol>
4. RNA-seq Analysis
<ol>
	<li>From the total RNA stock, make up 21 &#181;L of 100 ng/&#181;L RNA in RNase-free water. Use 1 &#181;L of this 21 &#181;L to test the RNA concentration as above. Submit the remaining 20 &#181;L (2 &#181;g) total RNA to an external sequencing center for mRNA enrichment, strand-specific library preparation (with eight or more barcodes for multiplexing) and sequencing (see Materials Table). Alternatively, locally perform mRNA enrichment and library preparation, and then submit the library for sequencing.</li>
	<li>Pool eight or more multiplexed samples and sequence in one lane of a next-generation sequencer.</li>
	<li>Download the FASTQ file containing all of the sequence reads and the corresponding index reads. Also, download the text file containing the multiplex barcodes. 
	NOTE: The index reads may be present in a separate FASTQ file. Place all of these files into one directory.</li>
	<li>Place the shell script provided here, &#34;fastq_pipeline.sh&#34;, in the same directory. Edit this script as appropriate for the experiment and the computer directories. 
	NOTE: this script contains extensive annotations explaining the steps. In brief, the script quality trims the reads, maps the reads to the genome, and generates a tab-delimited file containing the number of reads per gene for each sample.</li>
	<li>Deposit the FASTQ files and raw read count data into NCBI&#39;s Gene Expression Omnibus before publication 27. Follow the instructions for &#34;Submitting high-throughput sequence data to GEO&#34;: https://www.ncbi.nlm.nih.gov/geo/info/seq.html.</li>
	<li>Import the read count data into the R statistical environment and perform statistical analysis, using the R script provided here, &#34;time_course_script.R&#34;. Edit this script as appropriate for the experiment and the computer directories. 
	NOTE: This script contains extensive annotations explaining the steps. In brief, this script imports the read data, normalizes the reads, identifies genes that change significantly during the time course, performs PCA analysis, and graphs the expression of selected genes.</li>
</ol>

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J., Rine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Role+for+Sterol+Levels+in+Oxygen+Sensing+in+Saccharomyces+cerevisiae.">A Role for Sterol Levels in Oxygen Sensing in Saccharomyces cerevisiae.</a> Genetics. 174 (1), 191-201 (2006).</li><li>Meena, R. C., Kumar, N., Nath, S., Chakrabarti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+Recombination+is+Activated+at+Early+Time+Points+Following+Exposure+to+Cobalt+Chloride+Induced+Hypoxic+Conditions+in+Saccharomyces+cerevisiae.">Homologous Recombination is Activated at Early Time Points Following Exposure to Cobalt Chloride Induced Hypoxic Conditions in Saccharomyces cerevisiae.</a> Ind. J. Microb. 52 (2), 209-214 (2012).</li><li>Snoek, I. S. I., Tai, S. L., Pronk, J. T., Yde Steensma, H., Daran, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+Snf7p+and+Rim101p+in+the+transcriptional+regulation+of+TIR1+and+other+anaerobically+upregulated+genes+in+Saccharomyces+cerevisiae.">Involvement of Snf7p and Rim101p in the transcriptional regulation of TIR1 and other anaerobically upregulated genes in Saccharomyces cerevisiae.</a> FEMS Yeast Res. 10 (4), 367-384 (2010).</li><li>Ellahi, A., Thurtle, D. M., Rine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chromatin+and+Transcriptional+Landscape+of+Native+Saccharomyces+cerevisiae+Telomeres+and+Subtelomeric+Domains.">The Chromatin and Transcriptional Landscape of Native Saccharomyces cerevisiae Telomeres and Subtelomeric Domains.</a> Genetics. 200 (2), 505-521 (2015).</li><li>Collart, M. A., Oliviero, 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=Preparation+of+yeast+RNA.">Preparation of yeast RNA.</a> Curr. Prot. Mol. Biol. , (2001).</li><li>Blankenberg, D., 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=Manipulation+of+FASTQ+data+with+Galaxy.">Manipulation of FASTQ data with Galaxy.</a> Bioinf. 24 (14), 1783-1785 (2010).</li><li>Martini, P., 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=timeClip:+pathway+analysis+for+time+course+data+without+replicates.">timeClip: pathway analysis for time course data without replicates.</a> BMC Bioinf. 15, (2014).</li><li>Oh, S., Song, S., Grabowski, G., Zhao, H., Noonan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+series+expression+analyses+using+RNA-seq:+a+statistical+approach.">Time series expression analyses using RNA-seq: a statistical approach.</a> BioMed Res. Int. 2013, 1-16 (2013).</li></ol>

The authors have nothing to disclose.

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 <sup class="xr...

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 5,6,7,8,9,10,11,12. Recently, RNA-seq was used to characterize gene expression changes over time during hypoxia 13. Here, the experimental details are presented and discussed.
Hypoxia can be achieved in various ways, each producing a different level of O2. Here, hypoxia was established by continuously flowing ultra-high-purity N2 into flasks, which lowers [O2] dissolved immediately with reproducible kinetics 10. It is possible that there are some O2 molecules present that contribute to metabolism and gene expression but this environment is considered very close to anaerobic. In the absence of O2, yeast cells cannot biosynthesize heme, ergosterol and unsaturated fatty acids 4,12,14. Thus, previous studies have included these metabolites when growing yeast without oxygen 5,10,15. However, many hypoxic responses are mediated by depletion of these metabolites and thus replenishing them reverses the hypoxic gene expression responses 12,16. In order to mimic natural hypoxia, these metabolites were not added to the media. In the short time that cells were exposed to hypoxia without the presence of these essential metabolites, there was no noticeable increase in cell death (data not shown) nor a prolonged stress response 13.
The response is also dependent upon the strain and its genotype. Especially important are the alleles of the known regulators of hypoxic responses 2. The S288C strain background is highly desired so that results can be compared to the other genomic studies performed with this strain. However, S288C contains a partial loss-of-function allele of the HAP1 gene 17, a transcriptional regulator critical for the hypoxic response. This allele was repaired in S288C using a wildtype copy from the &#931;1278b strain background 11.
Gene expression is highly dependent upon the cellular environment. Thus, when performing genome-wide mRNA analysis, it is important to maintain a constant environment while varying another parameter such as time, stimulus, or genotype. To achieve highly reproducible results, consider these three practices for the study and all of its biological or technical replicates. First, the same experimenter(s) should carry out the study, since technical practices may vary across experimenters. Second, the same batch of ingredients should be used in the growth media as each batch has a slightly different composition that can affect gene expression. Third, to minimize cell cycle effects, each time point should consist of asynchronous cells in the mid-log phase of growth (1 - 2 x 107 cells/mL).
When characterizing a complex response like the gene expression response to hypoxia, a time course is advantageous for determining the kinetics of various events. Specific time points should be chosen that will capture the major changes of the response. In this study, time points between 0 and 4 h were observed, because past experiments revealed widespread changes in gene expression during this period 13.
To measure global gene expression, RNA-seq was used 18,19. This method uses next-generation sequencing to determine the relative abundance of each gene&#39;s transcript. Compared to DNA microarray analysis, RNA-seq exhibits higher sensitivity (to detect less abundant transcripts), a greater dynamic range (to measure greater fold changes) and superior reproducibility (to accurately follow gene expression over time). Typically, most cellular RNA is ribosomal RNA so many methods have been developed to enrich for specific RNA species 20. Here, poly-T beads were used to purify poly-A-containing mRNA transcripts, though the various commercially- available rRNA depletion kits could also be effective in mRNA enrichment.
Here, the S. cerevisiae gene expression response to hypoxia was characterized. Cells were exposed to hypoxia and then sampled at eight time points (0, 5, 10, 30, 60, 120, 180 and 240 min). To confirm reproducibility and to identify statistically changed transcripts, three biological replicates were performed. RNA was extracted by mechanical disruption and column purification, and then processed for RNA-seq analysis. The post-sequencing pipeline is described and programming scripts are provided that allow exact replication of the analyses performed. Specifically, Trimmomatic 21, TopHat2 22, HTseq 23, the R statistical environment 24, and the DESeq2 package 25 were used to process the RNA-seq data and to identify 607 genes that change significantly during hypoxia. Principal component analysis (PCA) and gene expression of the replicates indicated the reproducibility of the technique. Clustering and heatmaps revealed wide-ranging expression kinetics, while gene ontology (GO) analysis showed that many cellular processes, like aerobic respiration, are enriched in the set of oxygen-regulated genes.

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

measuring mrna levels over time during the yeast s. cerevisiae hypoxic response

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.

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...

Watch this Scientific Journal Video about Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response at JoVE.com

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

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.

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

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

measuring-mrna-levels-over-time-during-yeast-s-cerevisiae-hypoxic

Universidad San Sebastian

Research

JoVE Journal

Genetics

8.2K Views.  Rowan School of Osteopathic Medicine. 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.

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

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Biochemistry

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Biology

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

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

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Chapters in this video

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Microarray Analysis for Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth