Getting ready for RNA-seq cell/RNA preps

The RA's missing notebook hasn't turned up, so I don't have her notes of how she prepared the samples for the RNA-seq analysis. Luckily the main procedures are ones she used in many experiments and are described in her earlier notebooks and in an email she sent me.

The basic procedures:

Collecting samples:

1. Grow cells to desired state in rich medium (sBHI) or competence medium (MIV).
2. 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.
3. Mix 2 x 1 ml with 0.25 ml 80% glycerol and freeze at -80°C. (for later competence assays).
4. Pellet RNAprotect cells and freeze at -80°C.

Preparing RNAs:

1. Thaw cell pellets
2. Use Qiagen RNA prep kit.  We have lots and can get more quickly through LSC Stores.
3. Don't use the DNase step.
4. Elute in 40 µl H20.
5. Measure concentration of 1 µl with Nanodrop.
6. Run 4 µl in a 1% agarose TAE gel at 60V.

Treat to remove DNA:

1. Use volume containing 1 µg RNA (using RNA concentration from Nanodrop)
2. Use Ambion Turbo DNase 
3. Use protocol in RA's notebook #1 (not missing); 2 x 30 min incubations

Check RNA quality (and concentration?):

1. 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.

Treat to remove rRNA:

1. Use Ribo-Zero kit to remove the rRNA from each sample.

Check RNA quality (and concentration?) again:

1. 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.

Samples:
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.