RNA-seq progress, problems and plans

I've been growing the cell preps for the RNA-seq analysis, as shown in the planning figure below.

I did Day C's cultures, freezing 1 ml of cells for later transformation testing if needed, and 2 ml of cells for RNA purification.  The first snag was the invisibility of the cells!  Following instructions from the former RA, I mixed 2 ml cells from each of the first samples with 4 ml of the magic 'RNAprotect' reagent, let the mixture sit for 5 min at room temperature, and spun down the cells (2 ml of mixture in each of three 2 ml tubes) in our mini-spin microcentrifuge.  The plan was to discard the liquid and freeze the cell pellets at -80 °C, for later RNA preps.  But there was no visible cell pellet!  This amount of cells typically forms a small but easily visible pellet when centrifuged, but the bottoms of the plastic tubes looked perfectly clean.

I spun the tubes again - still no pellet.  So I pretended a pellet was present, discarded the liquid, and froze the apparently empty tubes anyway.  I checked the RNAprotect instruction booklet which reassuringly said that sometimes the pellet might be invisible, but I also contacted the RA, who said that her preps had given clearly visible pellets.  Later I thawed out one tube and did a wilful-suspension-of-disbelief RNA prep using the RNeasy kit, which produced the same concentration of (high-quality) RNA as the RA's original preps.  So I'm now assuming that there are invisible cell pellets in all the tubes, and I've done Day D's preps.

The RNAeasy kit also had some surprises, a solution that was supposed to be clear (or with a bit of particulates that could be removed by centrifugation), instead was very cloudy, and centrifuging it raised the cloudy material to the top (as a diffuse scum) rather than pelletting it.  I couldn't get rid of the scum (it just redispersed when I tried to pipette it), so I went on to the next step (adding 100% ethanol), which eliminated the cloudiness completely.  (Maybe this cloudiness came from the presence of too much residual RNAprotect in my tubes - because I couldn't see any pellet I didn't thoroughly drain the tubes before freezing them.)

I would have also tested the DNA-elimination step, which uses Turbo Dnase and a DNase-inactivator chemical.  But my brand new box of TurboDNase ($250) has gone missing.  I've searched the freezer a couple of times, and racked my brain in case I put it somewhere special for 'safekeeping', with no success. My next step is to go down to Stores and have them show me exactly what the TurboDNase box looks like, so I know I have the right search image.

I've also revised my plans for the cells I'll test.  As I wrote earlier, I'm only going to do one replicate of the ∆hfq mutant in MIV, since we really should use special precautions to avoid losing small RNAs from the prep (and maybe to do strand-specific sequencing).  Since we planned on three full lanes of Illumina sequencing, each 24-fold multiplexed, this change opens up space for 8 additional samples. Four of these will come from a MIV time-course using a crp or cya knockout strain (in Day E, which will be tomorrow).  This is an excellent control since it lets us identify all of transcripts dependent on the transcription factor CRP.  I'll also include both crp (or cya) and sxy knockout strains in the rich medium cultures on Day H; taking two time points for each will give the four additional cultures to complete the first two lanes of sequencing.