TITLE: RNA extraction with spin columns from yeast cells grown on 12-column deep well plates
AUTHORS: Jamie Auxillos, Rosey Bayne, Edward Wallace

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for yeast cell growth, lysis, and RNA extraction using silica spin columns. We use this to prepare RNA for RT-qPCR analysis from many strains at once. We have particularly used it on S288C-background </span><span style = "font-style:italic;">Saccharomyces cerevisiae</span><span>, containing a plasmid encoding a fluorescent reporter and a URA3 selection cassette, grown in SC-URA media. Then we use RT-qPCR to measure reporter gene expression compared to reference genes. In principle the protocol should work for similar strains/species and growth media, as long as the cell shape and cell walls are similar enough that the lysis method works.</span></div><br/><div class = "text-block"><span>Yeast strains are grown in parallel, in a 12 column deep well plate, on a shaking incubator allowing for easier strain handling. Yeast cells then are lysed by bead bashing with zirconia beads using the Precellys Evolution homogeniser. RNA is subsequently purified using a silica column. The RNA extraction method was a modification of the Quick-DNA/RNA miniprep kit from Zymo Research (D7001, </span><a href="https://www.zymoresearch.com/collections/quick-dna-rna-kits/products/quick-dna-rna-miniprep" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.zymoresearch.com/collections/quick-dna-rna-kits/products/quick-dna-rna-miniprep</span></a><span>). The key difference is that we find RNA binding buffer (R1013) is far more effective as a lysis buffer for our yeast samples than the buffer supplied with that kit. The protocol makes extensive use of Zymo Research columns and buffers, that can be purchased individually. Other manufacturers make similar columns and buffers or you can make your own, although we have not tested those (Yaffe et al. 2012; Jue et al., 2020).</span></div><br/><div class = "text-block">References:</div><div class = "text-block"><span>Jue, E., Witters, D. & Ismagilov, R.F. Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits. Sci Rep 10, 1940 (2020). </span><a href="https://doi.org/10.1038/s41598-020-58586-3" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/s41598-020-58586-3</span></a></div><div class = "text-block"><span>Yaffe, H., Buxdorf, K., Shapira, I.</span><span style = "font-style:italic;">et al.</span><span>LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues.</span><span style = "font-style:italic;">BMC Res Notes</span><span style = "font-weight:bold;">5,</span><span>45 (2012). https://doi.org/10.1186/1756-0500-5-45</span></div></div>

[STEPS]
?. [Harvesting and lysis of cells]
Take a screw cap tube (Sarstedt cat #12705493) and use a PCR tube to measure zirconia beads (BioSpec #11079105z) (note - fill the entire PCR tube with beads) . Add the zirconia beads into the screw cap tubes. Label each tube.
?. [Harvesting and lysis of cells]
Resuspend the pellet in the 12 column deep well plate with  of RNA binding buffer (Zymo cat no. R1013-2) and transfer them to the screw cap tubes containing the zirconia beads (keep on ice)
400 µl
?. [Harvesting and lysis of cells]
Transfer the tubes to the PreCellys (Bertin Instruments PreCellys Evolution) and run the EW Yeast protocol (this is a method). Chill tubes on ice for  . Repeat this step 5 more times (Total of 6 cycles)The Precellys Evolution EWYeast protocol is set to 3 cycles shaking at a speed of 6000 RPM, each with a duration of 10s and with a pause between each cycle of 10s.
Cell mixture tends to freeze when placed too long on ice. Thaw before putting it on the PreCellys. A frozen pellet can cause problem in the RNA extraction protocol and ultimately low yields.
?. [Harvesting and lysis of cells]
Transfer the tubes to a tabletop centrifuge and spin at .
Centrifuge: 12000 34, 90 s
The tubes tend to freeze and solidify when kept on ice for too long. Make sure that it is thawed before you transfer it to the centrifuge. A frozen pellet can cause problem in the RNA extraction protocol
DO NOT cool the centrifuge in this and all following steps. This causes problems in the RNA extraction process. Keep the centrifuge at room temperature.If you have a refrigerated centrifuge, set it to 22℃.
?. [RNA extraction]
Discard the Zymo IIICG and keep the flow through! Add  of 100% EtOH to the flow through on the collection tube and mix well by pipetting up and down at least 4x.
400 µl
Mix well in this step!
?. [RNA extraction]
Transfer the flow-through and ethanol mixture to a Zymo Spin IIC column (Zymo cat no. C1011) on a collection tube and spin at .
Centrifuge: 12000 34, 1 min
?. [RNA extraction]
Discard the flow-through and keep the column. Add  of DNA/RNA Prep Buffer (Zymo cat no. D7010-2) to the column. Spin at .
400 µl
Centrifuge: 12000 34, 1 min
?. [RNA extraction]
Discard the flow-through and add  of DNA/RNA Wash Buffer (Zymo cat no. D7010-3) to the column. Spin at .
600 µl
Centrifuge: 12000 34, 1 min
Make sure that you have added Ethanol to the concentrated DNA/RNA wash buffer, following the manufacturer's instructions. We recommend that this dluted wash buffer is not too old, as Ethanol and water evaporate at different rates, which disturbs the concentrations.
?. [RNA extraction]
Discard the flow-through and add  of DNA/RNA Wash Buffer to the column. Spin at .
400 µl
Centrifuge: 12000 34, 1 min
?. [RNA extraction]
Discard the flow-through spin again at .
Centrifuge: 12000 34, 1 min
?. [RNA extraction]
Transfer the Zymo IIC column to a clean 1.5 ml tube, labelled with your sample name as this will be used to store the RNA. Add of nuclease free water to the column (add this on the filter without touching the filter with your tip) and incubate at room temperature for  . Spin at  and discard the column. The RNA is now in the eluted flow-through.
30 µl
Centrifuge: 12000 34, 1 min
?. [Pre-culture growth]
Take a sterile 24 (deep) well plate (Star lab cat no. E2824-0000) and using a pipette controller, transfer  sterile media to each well of the 24 well plate according to the plate plan above (24 well plate plan in Step 2).
1 mL
?. [Plate plan]
Plan your 24 well culture plate layout - Subdivide strains into groups of 6 then fill out the strain names on the following pre-culture (24 well) plate plan to log the locations of each strain on the 24 well plateTemplate 24 well plate plan - Example 24 well plate plan -
Make sure you have media only wells to serve as a negative control!
By biological replicate (biorep), we usually mean single colonies of the same genetically identical stock, grown on the same agar plate.
?. [Pre-culture growth]
Using sterile P20 tips, inoculate the media in each well with a single colony of the appropriate strain according to the plate plan above (24 well plate plan in Step 2).
Use a new sterile tip for each strain - do not re-use tips for multiple wells or multiple strains.
?. [Pre-culture growth]
Seal the deep well plate with a gas permeable seal (Thermo cat #AB0718) and grow overnight in a shaking incubator set at   and 200 rpm.
30 °C
?. [Pre-culture growth]
After 16-20 hours, for each strain transfer   of the overnight culture to a cuvette then add of  media and mix well (1:10 dilution of the overnight culture) and measure the OD600 using a spectrophotometer.
100 µl
900 µl
16-20 hours is enough time for the culture to reach saturation.
?. [Culture growth from an OD600 of 0.2 to 0.5-0.7]
Take sterile 12 column deep well plates (4titude cat #PCR0244) (based on the plate plans above, we will need 2 for this example). Using a pipette controller (Eppendorf cat #4430000018), dispense  of media into each well of the 12 column deep well plate.
7 mL
?. [Pre-culture growth]
Calculate the amount of the overnight culture to add to 8 ml of sterile media to reach a starting OD600 of 0.2.
?. [Culture growth from an OD600 of 0.2 to 0.5-0.7]
Transfer the calculated amount of the overnight culture into their respective wells on the 12 column deep well plate, based on the amount calculated in step 8.
Change the pipette tip for each strain/biorep.
?. [Culture growth from an OD600 of 0.2 to 0.5-0.7]
Seal with a gas permeable seal and grow in a shaking incubator set at   and 90 rpm to an OD600 of 0.5 - 0.7.
30 °C
This should take ~3.5 - 4 hours.
?. [Culture growth from an OD600 of 0.2 to 0.5-0.7]
Take  of the diluted culture and measure their OD600 using a spectrophotometer (to check that their starting ODs are betwee 0.15 - 0.2).
1 mL
?. [Harvesting and lysis of cells]
Once the OD600 of the culture has reached between OD600 of 0.5-0.7, pellet the cells on the 12 column deep well plate by centrifugation at .
Centrifuge: 3000 34, 3 min
?. [Culture growth from an OD600 of 0.2 to 0.5-0.7]
After ~3.5 - 4 hours, measure the OD of each culture by transferring  of the culture using a spectrophotometer.
1 mL
?. [Harvesting and lysis of cells]
Using an aspirator, remove the media in each well. Avoid touching the pellet.
DO NOT decant! The pellet gets loosened and increases the chance of cross contamination between wells.
When using the aspirator, change the tip for each well to avoid cross contamination.Try to remove as much media as possible. Leaving a bit of media in the well can affect the RNA extraction efficiency.
**This is a possible pause step - The pellets in the deep well plate can be frozen at . It is better to freeze it this way than in RNA binding buffer with the zirconia beads.
?. [Harvesting and lysis of cells]
Transfer supernatant to a Zymo Spin IIICG column (Zymo cat no. C1006) on a collection tube (Label tubes). Spin the Zymo Spin IIICG containing the supernatant at . (This step binds DNA to IIICG column).
Centrifuge: 12000 34, 1 min
?. [RNA extraction]
Measure the concentration of the RNA using a microvolume spectrometer (e.g.nanodrop). Verify the quality of the RNA by running samples on the fragment analyzer.
Expected result - The RNA concentration should be between 1000-2000 ng/ul.A gel or capillary electrophoresis trace should show 2 clear ribosomal RNA bands. See RNA: a laboratory manual (Rio, Ares, Hannon, Nilssen) for more on RNA quality.
Store the RNA in   .For long-term storage or to send to a sequencing facility, it is often recommended to store in low-binding tubes and in low TE buffer.
?. [Plate plan]
Plan your 12 column culture plate layout - Subdivide the strains into groups of 12 then fill out the strain names on the following 12 well plate plan to log the locations of each strain on plate.Template 12 column deep well plate plan - Example 12 column deep well plate plan -
Again, make sure you have media-only wells!
?. [Grow single colonies of yeast]
Streak out yeast from glycerol stocks to single colonies on petri dishes poured with solid media that is appropriate for your experiment, and grow as desired. We usually grow for 2 days () at . To minimize experimental variability, it is best to use fresh plates, i.e. that have never been stored in a fridge.
30 °C