I'm developing libraries for RNA-seq on the Illumina platform. I have a couple of questions:
1.) After you have made double stranded cDNA from mRNA should you see a bump on a bioanalyzer RNA Pico chip? I see a small bump in the fragmented RNA after I precipitate it, but not when I run a sample of the ds cDNA. I'm assuming that this is because the ds cDNA is substantially more dilute than the fragmented RNA. Is this a 'normal' result?
2.) I'm using Ambion's RNA fragmentation kit to fragment my RNA. Has anyone had success with this kit? If so what were your fragmentation parameters?
3.) At what sort of starting quantities of total RNA have people had success generating libraries for RNA sequencing? I'm using ~ 10 ug of total RNA.
Thanks in advance for your help!
1.) After you have made double stranded cDNA from mRNA should you see a bump on a bioanalyzer RNA Pico chip? I see a small bump in the fragmented RNA after I precipitate it, but not when I run a sample of the ds cDNA. I'm assuming that this is because the ds cDNA is substantially more dilute than the fragmented RNA. Is this a 'normal' result?
2.) I'm using Ambion's RNA fragmentation kit to fragment my RNA. Has anyone had success with this kit? If so what were your fragmentation parameters?
3.) At what sort of starting quantities of total RNA have people had success generating libraries for RNA sequencing? I'm using ~ 10 ug of total RNA.
Thanks in advance for your help!
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