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 can=me 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.
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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 can=me 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. On other days the frozen viable cells will be thawed and transformed to check that they have the expected level of competence - I can probably do a lot in one experiment.
Ater all the samples have been collected, he 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, then treated to remove the bulk of the ribosomal RNA (this costs a lot too, 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).