Hi Lizex,

> I've started analyzing my RNA-Seq data for two time points: Day0 and Day4 for 
> control and treated. I've done aligning the data to the reference genome 
> using Tophat. I've removed duplicates from the data sets. Could somebody 
> please tell me, how important is it to remove duplicates and how will it 
> influence my results if I don't remove?

This depends on whether you are removing duplicates in your fastq data and/or 
multi-mapping reads either using Tophat or post-processing steps. In any case, 
this approach that will affect quantitation outputs from Cufflinks and likely 
transcript assemblies as well.

> I want to start with Cufflinks all the way through to Cuffdiff. Where do I 
> start since there are just so many options (in the manual) to choose from? 
> What do I look for?

Here's a tutorial that will help you get started with RNA-seq analysis: 

Galaxy makes it easy to experiment with different parameter values, so you'll 
want to read the Cufflinks/compare/diff manual and adjust parameters that are 
relevant to your data:


In general, RNA-seq studies look at (a) transcripts assembled; (b) expression 
values; and (c) differential expression estimates.

Good luck,

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