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First try in understanding RNA-seq
Dear all!!
It is a first time, I am going to set up RNA-seq experiments in our lab. We wonder to utilize two-paired RNA-seq experiment with total RNA prep from yeast. While analyzing present articles in this field, I was focused on next question:
1) how to prepair cDNA library
2) how the Illumina GA works
3) how to analyze results
Does some global overview of these questions exist in the Internet? I mean, the web-source, where you can find user-protocols at each step, useful programs, suggestions, and ets?
Then, to address the first question, I have found some published methods (http://www.nature.com/nprot/journal/....2009.229.html for instance), but in any case I have some question riased.
How do you usually do the size selection technically? I mean, there is special equipment exist, or it is simply done by cutting pieces of the gel?
What alternative approaches exist in size selection?
Secondly, I cannot understand totaly, what the reason to do PCR enrichment of ligated products? Maybe I am missing something, but after that it is uniformally assumed that RNAseq is quantitive, but how it could be, If you utilize PCR?
In any case, I have found the article about FRT-seq method (http://www.nature.com/nmeth/journal/...meth.1417.html), where it is shown rather good results of merging cDNA -sinthesis on the flow cell of Illumina GAII. Results are really great.
How do think, what the reason of not huge popularity of this approach? What the cautions could exist utilizing this method?
It is a first time, I am going to set up RNA-seq experiments in our lab. We wonder to utilize two-paired RNA-seq experiment with total RNA prep from yeast. While analyzing present articles in this field, I was focused on next question:
1) how to prepair cDNA library
2) how the Illumina GA works
3) how to analyze results
Does some global overview of these questions exist in the Internet? I mean, the web-source, where you can find user-protocols at each step, useful programs, suggestions, and ets?
Then, to address the first question, I have found some published methods (http://www.nature.com/nprot/journal/....2009.229.html for instance), but in any case I have some question riased.
How do you usually do the size selection technically? I mean, there is special equipment exist, or it is simply done by cutting pieces of the gel?
What alternative approaches exist in size selection?
Secondly, I cannot understand totaly, what the reason to do PCR enrichment of ligated products? Maybe I am missing something, but after that it is uniformally assumed that RNAseq is quantitive, but how it could be, If you utilize PCR?
In any case, I have found the article about FRT-seq method (http://www.nature.com/nmeth/journal/...meth.1417.html), where it is shown rather good results of merging cDNA -sinthesis on the flow cell of Illumina GAII. Results are really great.
How do think, what the reason of not huge popularity of this approach? What the cautions could exist utilizing this method?