Field of Science

Plans for RNA-seq analyses

I should have posted this after last week's lab meeting but am only now getting to it.  I sensibly took snapshots of the whiteboard at the end of that lab meeting, so I could check what we'd decided.

The issues:  We have several Haemophilus influenzae mutants whose gene-expression profiles we want to examine, either during competence development in the MIV starvation medium or during normal growth in the rich culture medium sBHI.  For most of these (i) we want samples from several timepoints over a few hours, (ii)we want wildtype controls done in the same experiment, and (iii) we want three replicate samples from experiments done on different days.  And it would be nice to have multiples of 24 samples, since the kits and sequencing are most efficient with that.

That's a lot of constraints , but we came up with a plan that meets them all:  The first two days are the samples that have already been prepared and sequenced; the other 6 days are for me to generate the samples.

The samples will consist of viable cells frozen in glycerol (one or two 1.5 ml tubes), and duplicate pellets of cells that have been briefly incubated with a RNA-protection reagent to stabilize their RNA.  Later the frozen viable cells will be thawed and transformed to check that they have the expected level of competence - I can probably do all of them in a few days.

After all the samples have been collected, the RNA-prep pellets will be thawed and the RNA isolated using a kit.  After a quick check of RNA concentration and size, the contaminating DNA is removed by treatment with Turbo-DNase.

All the samples are then checked for concentration and quality using a special something (the post-doc recommends, using equipment in another lab), then treated to remove the bulk of the ribosomal RNA (this kit costs a lot, I think $200 per sample), then rechecked for concentration and quality using the special something.

Finally the samples are ready to be made into multiplexed sequencing libraries (expensive service on campus) and then sequenced (another service on campus).

To order (first check what we have on hand):
  •  RNA protection reagent for 72 samples
  • RNA prep kits for 72 samples
  • Turbo-DNase for 72 samples
  • Does the 'special something' require special reagents?
  • rRNA-removing kits for 72 samples
The reasons for doing these experiments are described briefly in this post.  

Because my hydroxyurea experiments found that cell doubling is substantially faster when cultures are at very low cell density, for the first samples in experiments F. G and H I'll use large volumes of cells at a lower cell density than the usual OD600=0.2.  Getting the right density will be a bit tricky since we can't use OD600 to check densities of very dilute cultures.  In the table above I said I'd use an OD600 of 0.02, but I'll start by growing cells from a lower density to about this OD and then diluting them 16-fold so they'll have another four doublings before I sample them.  The hydroxyurea-experiment cultures were much more dilute than this, but I need to balance culture density and culture volume so I'll have enough cells for the RNA extractions.  With this plan I'll be collecting cells from about 2 x 250 ml of culture for the first samples, and combining the remaining volumes in the two flasks for the later samples.

Other experiments to do:  We need to sequence the genome of strain RR753, to identify candidate mutations that cause its hypercompetence.  Before doing this I should recheck it and the backcross strain I made - if they both are hypercompetent we'll want to sequence both.

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