Field of Science

RNA-seq progress

I've collected and frozen all the 24 samples for our make-up RNA-seq run. (not the Trizol-prep ones - they've been deep-sixed).  And this morning the co-op tech learned to prep RNA from each sample. She's done the first 4, and will do the rest over the next couple of days.

The next steps are:

  1. Complete the PCR tests of strain genotypes and the analysis of transformation frequency data. She's still working on the PCR tests, but so far everything looks OK.
  2. Check the RNA concentration using the Nanodrop
  3. Run aliquots of the samples in a gel to check integrity of the rRNA bands (surrogate for integrity of the mRNA).
  4. Treat 5 µg of each sample with DNA-free.  We found our stock from last year, and there's still enough to treat all our samples.
  5. The former RA says we can take the DNA-free-treated samples directly to the RiboXero ste; we don't need to first do a 'clean-up' step with the RNeasy Minelute kit spin-columns.
  6. Treat an aliquot of each sample with RiboZero.  We will use only half as much RNA as recommended, and only 1/4 as much of the other reagents (in 1/4 of the recommended volume, of course). This will let us treat 24 samples with a 6-treatment RiboZero kit.
  7. Give the samples to the former RA in her new lab for library preparation and sequencing.

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