We are about to embark on an RNAseq project looking at the effects of a null mutant mouse lacking an RNA binding protein that affects mRNA stability/translation and splicing. So, we want to explore both mRNA levels as well as mRNA exon composition in mutants vs wild-type.
1) Machine
In theory, we have access to 454, SOLiD, Illumina, although the latter is best established in the facility. Read length is obviously important for a splicing analysis (an argument for 454) but a paired end on Illumina would probably also do the trick?
2) RNA isolation & library
We were thinking of using a standard RNeasy plus purification, which does not catch RNAs under 200nts. Any downsides you can think? Or a different isolation you think superior for our purpose?
Followed by library generation in the facility which as far as I understand is a typical oligoT primed cDNA generation. RNA quantity is not an issue, so we don't need error-prone amplification.
3) Type of read
Should we go for maximal read length & paired end or is less good enough?
What number of total read should we aim for to be able to do the analyses we want to do?
Of course, long, paired, and more reads will give us better data but what's a good minimum starting point?
Would be great to hear your opinions.
Regards, j
1) Machine
In theory, we have access to 454, SOLiD, Illumina, although the latter is best established in the facility. Read length is obviously important for a splicing analysis (an argument for 454) but a paired end on Illumina would probably also do the trick?
2) RNA isolation & library
We were thinking of using a standard RNeasy plus purification, which does not catch RNAs under 200nts. Any downsides you can think? Or a different isolation you think superior for our purpose?
Followed by library generation in the facility which as far as I understand is a typical oligoT primed cDNA generation. RNA quantity is not an issue, so we don't need error-prone amplification.
3) Type of read
Should we go for maximal read length & paired end or is less good enough?
What number of total read should we aim for to be able to do the analyses we want to do?
Of course, long, paired, and more reads will give us better data but what's a good minimum starting point?
Would be great to hear your opinions.
Regards, j
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