The main advantage of this technique is that it reflects RNA polymerase II activity by measuring newly-synthesized mRNA which are not influenced by RNA degradation. This protocol was originally described using microarray hybridization, but can easily be adopted to high-throughout sequencing of newly-transcribed mRNA. Demonstrating this procedure will be Tiago Baptista, a former grad student from our laboratory.
After inoculating the S.cerevisiae cells, grow them overnight at 30 degrees Celsius with constant agitation at 150 RPM. The next day, measure the optical density at 600 nanometers. Dilute the culture to an OD600 of approximately 0.1 in 100 milliliters of YPD medium.
Grow up the cells until the OD600 is around 0.8. After this, measure the OD600 of the S.pombe overnight culture. Dilute this culture to an OD600 of approximately 0.1 in 500 milliliters of YAS medium and let it grow up until the OD600 is approximately 0.8.
First, prepare a fresh solution of two molar four-thiouracil. Keep the prepared solution at room temperature and protected from light. Add the four-thiouracil solution to the S.cerevisiae and S.pombe cultures such that the final concentration is five millimolar.
Incubate the S.cerevisiae and S.pombe cultures at 30 degrees Celsius and 32 degrees Celsius respectively with constant agitation for six minutes. After this, remove a small aliquot of each culture and count the cells using either an automatic cell counter or a Neubauer chamber. Centrifuge at 2, 500 times g and four degrees Celsius for five minutes to collect the cells.
Discard the supernatant and wash the cells with ice cold one times PBS. Centrifuge again at 2, 500 times g and four degrees Celsius for five minutes. Next, resuspend the cells in five milliliters of ice cold one times PBS.
Mix the S.cerevisiae and S.pombe cells at a ratio of three to one. Centrifuge the mixed samples at 2, 500 times g and four degrees Celsius for five minutes. Then remove the PBS and flash freeze the cells in liquid nitrogen.
Store the sample at negative 80 degrees Celsius until ready to use. When ready to proceed, thaw the cells on ice for approximately 20 to 30 minutes. Use an adapted yeast RNA extraction kit to extract the RNA as outlined in the text protocol.
After this, transfer the filter cartridge to the final collection tube. Elute the RNA with 50 microliters of DEPC-treated RNase-free water that was preheated to 100 degrees Celsius. Centrifuge at 16, 000 times g for one minute.
Then use 50 microliters of preheated DEPC-treated RNase-free water to elute the RNA again to the same tube. Centrifuge at 16, 000 times g for one minute making sure that the entire sample volume has passed through the filter. If it has not, centrifuge again for a longer period of time.
When finished, quantify and check the purity of the sample using the appropriate equipment. Use DEPC-treated nuclease-free water to adjust the concentration of the previously acquired RNA to two milligrams per milliliter. Aliquot 200 micrograms of total RNA and heat it at 60 degrees Celsius for 10 minutes.
Immediately chill it on ice for two minutes. Add 600 microliters of DEPC-treated RNase-free water. Add 100 microliters of biotinylation buffer and then add 200 microliters of biotin HPDP.
If the biotin HPDP solution precipitates out of the solution, increase the volume of DMSO or DMF up to 40%of the reaction volume as outlined in the text protocol. Protect the sample from light and incubate it at room temperature with gentle agitation for three hours. After this, add an approximately equal volume of chloroform to the tubes and mix vigorously.
Centrifuge at 13, 000 times g and four degrees Celsius for five minutes. Next, carefully transfer the upper phase into new two milliliter tubes. Add an amount of five molar sodium chloride equal to approximately 1/10 of the sample volume.
Then mix the sample. First, heat the biotinylated RNA at 65 degrees Celsius for 10 minutes. Then chill the samples on ice for five minutes.
Add 100 microliters of Streptavidin-coated magnetic beads to the biotinylated RNA. Incubate at room temperature with slight shaking for 90 minutes. Place the columns provided with the kit in the magnetic stand.
Next, add 900 microliters of room temperature washing buffer to the columns. Apply the 200 microliter bead and RNA mixture to the columns. Collect the flow-through in 1.5 milliliter tubes and then apply it again to the same magnetic column.
Keep this flow-through if necessary as it represents the unlabeled RNA fraction. To begin RT-qPCR validation of the different fractions, first synthesize cDNA as outlined in the text. Then amplify the cDNA by real-time qPCR using a standard protocol.
The measured levels of transcripts in fractions purified from wild-type cells cultured with and without four-thiouracil confirmed this procedure specifically purifies labeled RNA. Analysis of the spt20 delta strain reveals that the quantity of total steady-state RNA is largely unchanged or is only mildly reduced for the tested genes. Similar results are seen for strains deleted for bre1.
However, analysis of newly-transcribed RNA in the spt20 delta strain reveals a three to five-fold decrease in mRNA synthesis for all tested genes. Meanwhile, the loss of bre1 leads to a more discreet but still visible decrease. Upon inducible depletion of spt7, a structural subunit of the saga complex, newly-transcribed mRNA levels are reduced to an extent similar to the deletion strain.
When total RNA levels are measured by genome-wide analysis, only a few genes are seen to have their expression levels altered upon deletion of spt20 either by up regulating or down regulating. However, analysis of the newly-transcribed RNA reveals that the levels of over 4, 000 genes are significantly reduced at least two-fold upon deletion of spt20 which suggests a global positive effect of saga on RNA polymerase II transcription in budding yeasts. Though this method can provide insight in transcription in Saccharomyces cerevisiae, it was only applied with slight variations to By using this protocol, we could detect a global decrease in mRNA synthesis which was compensated by a global decrease in mRNA degradation.
