The basic procedures:
- Grow cells to desired state in rich medium (sBHI) or competence medium (MIV).
- Mix 2 ml with 4 ml RNAprotect reagent (Qiagen); leave 5 min at RT. We have 100 ml and can get more quickly through LSC Stores.
- Mix 2 x 1 ml with 0.25 ml 80% glycerol and freeze at -80°C. (for later competence assays).
- Pellet RNAprotect cells and freeze at -80°C.
- Thaw cell pellets
- Use Qiagen RNA prep kit. We have lots and can get more quickly through LSC Stores.
- Don't use the DNase step.
- Elute in 40 µl H20.
- Measure concentration of 1 µl with Nanodrop.
- Run 4 µl in a 1% agarose TAE gel at 60V.
- Use volume containing 1 µg RNA (using RNA concentration from Nanodrop)
- Use Ambion Turbo DNase
- Use protocol in RA's notebook #1 (not missing); 2 x 30 min incubations
- Use the 'Bioanalyzer' (high-tech equivalent of gel electrophoresis) to check the size distribution of the RNAs in each prep. Expect to see 2 strong rRNA peaks.
- Use Ribo-Zero kit to remove the rRNA from each sample.
- Use the 'Bioanalyzer' (high-tech equivalent of gel electrophoresis) to check the size distribution of the RNAs in each prep. Expect to see no rRNA peaks.
Here's the chart showing the MIV-competence samples I had planned. I'm only going to do one set of the ∆hfq strain now, because our procedures aren't optimized for small RNAs (poor recovery and no strand information). One of our summer-Honours students will be working on this mutant, and he can take my preliminary data and use it to help design an optimized RNAseq procedure. So I think on Day C I'll only do the three strains (sxy-, ∆659 and ∆6759/660), and if this goes smoothly scale up to four strains on Day D. Maybe on Day E I'll replace the ∆hfq strain with something else I want preliminary data about.