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[Drosophila] High throughput Q

sandra schulze via dros%40net.bio.net (by heterochromatic from gmail.com)
Thu Aug 8 15:02:15 EST 2013


People of Flyland,

I am trying to write a coherent description of a translation state
analysis, using RNA seq  for analysis. What I am looking for is advice
from a  technician/student/postdoc/PI who has had practical experience
with this method.

For RNA-seq, I'd like to know the following:

1. How much RNA is typically extracted from how many fractions of
polysomes? Is RNA pooled from fractions containing 1-5 ribosomes, and
then another pool from 6 or more OK? If each sample is one rep, are 3
reps enough?
2. What kits are typically used to get the RNA out of the polysome fractions?
3. Is the RNA extracted in #1 and 2 above enough for how many RNA-seq samples?
4. which platform - for eg. with Illumina, is the benchtop MiSeq
enough? Or would another platform be better?
5. How much sequence from a given sample (polysome fraction or pooled
fractions) is required to reach saturation? I.e., How much sequence is
needed per sample to be sure you've got everything sequenced?
6. How are the sequences normalized? Do you spike the samples with
some known synthetic control? Where does that come from?
7. Is there a bioinformatics pipeline specifically for Drosophila
sequences that is accessible for analysis?

You can see these questions are pretty green. Any help appreciated.

Thanks,

Sandra
Sandra Schulze
Associate Professor
Dept Biology
Western Washington University
Bellingham WA 98225



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