Tuesday, April 7, 2015

Back to planning the new RNA-seq samples

I just taught my last class of the term, so now I can get back to experiments.  The highest priority is preparing the new samples for RNA-seq analysis.  The preliminary planning is described in the previous post, which has a table summarizing both the runs we've done and the provisional plans.  Here it is again.

The plan is to get the RNA samples for these TO DO runs finished this month, before our Co-op tech finishes her work term and leaves us.

First step:  Decide what samples to collect and check what supplies we'll need to order (RNA-Protect, RNeasy kit, Trizol, DNA-free, RNeasy MinElute cleanup kit, Ribo-zero?).  We may collect a few more samples than the 24 we will be able to sequence.]

Second step:  Streak out all the strains.  Make especially sure that the antx and toxx strains are the unmarked ones, by checking their StrR and competence phenotypes.  In fact, check the StrR and competence phenotypes of everything.  Maybe use PCR to check the genotypes?

Third step:  Inoculate an overnight culture of each strains, Grow each strain as specified and collect RNA-protect treated cell pellets (store at -80 °C).

Fourth step:  Purify RNA from all samples except the 'kw20 Trizol' ones using the Qiagen RNeasy kit.  Check nucleic acid concentration using the nanodrop.  

Fifth step:  Purify RNA from the 'kw20 Trizol' samples using Trizol.  Check nucleic acid concentration using the nanodrop.

Sixth step:  Treat each RNA sample with DNase ('DNA-free'?).  Repurify using the Quigen RNeasy cleanup kit.  Check RNA concentration using the nanodrop.  If we have more than 24 samples, decide which ones to not sequence.

Seventh step:  Give samples to Sunita (?), who will treat them with Ribo-zero using half as much RNA and 1/4 as much Ribo-zero as recommended (thus using only a 6-sample kit rather than a 24-sample kit).  (Or maybe we will do this step ourselves.)

Eighth step:  Sunita will prepare these samples for sequencing.

So, first step.

Which samples to collect?  Has anything changed?  Well, before the sabbatical visitor left she did isolate a new rpoD mutation causing hypercompetence.  (Here I'll refer to it as rpo2).  Should we include it in the RNA-seq samples?  If we do, we'll have to cut out something else.  Its competence phenotype (from a sBHI tiome course) is very similar to that of the rpoD mutant we are including, so I think we won't include this one.

What do we need to order?

  • RNA-Protect?  We have 86 ml of RNA-Protect; this is enough for 21.5 2 ml samples.  Rather than buying more RNA-Protect ($278 for 200 ml), we'll just reduce our sample sizes slightly (1.6 ml) so we have enough for all of them.  This should still give us plenty of RNA.
  • Qiagen RNeasy purification kit:  We have a nearly complete kit with 47 of its 50 columns left.
  • Trizol:  We have a nearly full bottle of Trizol, though it's at least several years old.  It's been stored in the dark at 4 °C so it's probbly still OK.  A new bottle would cost about $230, so if we decide ours is too old we'll just scrounge the few ml we'll need.
  • RNeasy MinElute cleanup kit:  our old kit has 23 columns remaining, almost enough for one treatment of each of our smples (before Ribo-zero treatment).  But if we do the Ribo-zero work ourselves we'll need to also do a second cleanup after the Ribo-zero, so we'd need to buy another kit (about $400).
  • Ribo-zero:  If we do the treatment ourselves we'll need to order a 6-sample kit ($650).  However our collaborators (the former RA's new lab) might do this for us.  I'll check with her.

The tech is streaking out all the strains on plain sBHI plates.  We'll then restreak them on antibiotic plates to check their resistances, and confirm their transformation phenotypes when we grow them for the RNA preps.


  • KW20:  our wildtype strain (RR722, in freezer box 12): sensitive to all antibiotics, normal transformation.
  • HI0659 (antitoxin) knockout (unmarked) (RR3158, in freezer box 39): should be sensitive to all antibiotics, not transformable at all.
  • HI0660 (toxin) knockout (unmarked) (RR3159, in freezer box 39): should be sensitive to all antibiotics, normal transformation.
  • rpoD753 (RR753, in freezer box 16): resistant to streptomycin, moderately hypercompetent in sBHI.
  • hfq (marked deletion) (RR3187, in freezer box 40): resistant to spectinomycin, transformation down ~ tenfold.
  • crp::miniTn10kan (RR540, in freezer box 3): resistant to kanamycin and streptomycin, not transformable at all. 
  • sxy::miniTn10kan (RR648, in freezer box 8): resistant to kanamycin, not transformable at all.