Sample Prep for Microarray Analysis Microarray analysis requires high quality RNA extracted from cell culture

samples that are/were grown under stress conditions. The key to the process is speed: quickly harvesting the samples, resuspending them in a special buffer to stop RNA degradation, and the rapid RNA extraction (possibly at a later date). Each process is outlined below. We will be comparing the mutants to their respective parents under NaCl stress conditions to identify transcriptional perturbations. Sample Harvesting: 1. Pick two colonies for each strain (5 strains / day) and inoculate into M9 + glucose (0.5%) + tryptophan (50 ug/ml). 2. Grow overnight. 3. The next day, dilute 500 ul of the overnight cultures into separate flasks with 25 ml of M9 + 0.55 M NaCl + 0.5% glucose + 50 ug/ml. Use baffled flasks. a. 22.5 ml M9 b. 2.5 ml 5.45 NaCl-M9 stock c. 300 ul of glucose (40% stock) d. 185 ul of tryptophan stock 4. Load 5 ml of RNALater into 50 ml Falcon tubes (10 tubes total) a. RNALater prevents degradation of RNA, so we use it to preserve a snapshot of the transcriptome after the cells are harvested. 5. Grow until the cultures reach OD = 0.5-0.7 (mid-exponential phase). Always measure the actual OD of the cultures (no eyeballing). a. The mutants should reach this density after 8 hours of growth for the mutants, 10 hours for the 2xOriT parent, and 20 hours for the Delta parent. b. I highly recommend starting the Delta cultures late in the day (around 5pm) so they will be ready for harvesting around 1pm the next day. 6. Harvest the cells. a. Remove one flask from the shaker. b. Filter through a 0.45 um Nalgene filter with a removable membrane. c. Using tweezers, transfer the membrane to a falcon tube with RNALater. d. Resuspend cells by vortexing. 7. Freeze cells at -80 C until you are ready to extract the total RNA.

RNA Extraction: KIT!!! Prior to starting, make sure you have a falcon tube of 100% ethanol on hand. Make sure to only use that ethanol for RNA extraction experiments, as it is very easy to contaminate reagents with RNA degrading enzymes. 1. Pellet 3.0 ml of cells from each RNALater suspension in a microcentrifuge tube. a. If necessary, thaw the samples if you are removing them from the -80 C freezer. b. Place the samples back in the -80 C once you have the cell pellets that you need (if you have an appreciable amount left). 2. Resuspend each pellet in 600 ul of RNA Lysis buffer by vortexing. 3. Centrifuge the lysates for 1 minute @ 16,000g to remove cell debris. 4. Transfer 590 ul of lysate to a SpinAway Filter (yellow ring) in a collection tube. Centrifuge for 1 minute @ 16,000 g. Save the flow through. 5. Add 590 ul of 100% ethanol to each cleared lysate from step 4. Mix by pipetting up and down until nucleic acids are completely dissolved. 6. Transfer the mixture to Zymo-Spin IIICG column in a collection tube. Centrifuge for 30 seconds @ 16,000 g. Discard the flow though. 7. Prepare the DNase cocktail (amounts below are for 11 samples) a. 55 ul of DNase I b. 88 ul of Dnase reaction buffer c. 33 ul of DNase/RNase-Free Water d. 704 ul of RNA wash buffer 8. Add 400 ul of RNA wash buffer to each column and centrifuge for 30 seconds at 16,000g. Discard the flow through. 9. Add 80 ul of the DNase cocktail to each column. Incubate at room temperature for 15 minutes, then centrifuge for 30 seconds at 16,000g. 10. Add 400 ul of RNA prep buffer to each column and centrifuge for 30 seconds at 16,000g. Discard the flow-through. 11. Add 700 ul of RNA wash buffer to each column and centrifuge for 30 seconds at 16,000g. Discard the flow-through. 12. Add 400 ul of RNA wash buffer to each column and centrifuge for 2 minutes to ensure complete removal of the wash buffer. 13. Carefully remove the column from the collection tube. Inspect it for any residual wash buffer contamination; if the outside of any column looks wet, recentrifuge all of the columns for 1 more minute. 14. Transfer the columns to a RNase-free microcentrifuge tube. Add 50 ul of DNase/RNase free water to each column. Centrifuge for 30 seconds at 16,000g to elute RNA. 15. Take the eluted RNA and place it back in the corresponding column. Centrifuge again for 30 seconds at 16,000g to elute any residual RNA still bound to the column. 16. Transfer 3 ul of RNA to another labeled microcentrifuge tube. Proceed to quantification and qualification.

Quantification (concentration measurements) 1. Prepare the Qubit RNA BR fluorescent buffer for N+1 samples. a. (N+1) x 199 ul buffer b. (N+1) x 1 ul RNA dye 2. Vortex buffer+dye mixture, and aliquot 199 ul into N Qubit assay tubes labeled with sample IDs. 3. Add 1ul of RNA into each tube. Vortex briefly, then wait 1 minute. 4. Measure the RNA concentration using the Qubit. Qualification (gel electrophoresis) 1. Take 2 ul of each RNA sample and transfer it to a labeled microcentrifuge tube. 2. Add 2 ul of RNA loading dye (stored in the 4 C fridge) to each sample. 3. Transfer each sample to a TAE gel. Run at 135 V for 18 minutes 4. Image the gel. a. You should see two bands for each sample, one large one small. b. If you see a smear, the sample is degraded and must be re-extracted c. If you see nothing, the sample is also degraded (or the well was empty) 5. Save the image on the group drive and send it to both of us.