Seramag vs Dynabeads

[mapplebe]

Hello, I am trying to set up our lab's first RNAseq project (using Illumina single-reads), and was hoping I could get some advice.

The first is whether there is any significant difference in yield between Seramag and Dynabeads. From what I can tell, you can use 15 ul of seramag beads per prep (~$2/prep) or 100 ul of Dynabeads (~$20/prep). Most protocols I have found in papers have used Dynabeads, though the Illumina protocol uses Seramag. Is it safe to assume the extra cost is worth it?

Second is whether its easier to fragment at the RNA or cDNA stage. I am supposed to train undergrads to help with this project so I would prefer to get my mRNA to cDNA as early in the protocol as possible. Also, the transcript-end bias introduced by cDNA fragmentation isn't a concern for this project. However, reading through the discussion threads on here about cDNA fragmentation I've begun to suspect that RNA hydrolysis might cause fewer headaches? I may have access to a bioruptor, but other than that I would need to enzymatic cDNA fragmentation methods (DNAse or the NEB/Epicenter kits)

Thanks

[eab]

I don't know the differences between the two brands, but I am told by Invitrogen tech support that Dynabeads can be scaled down based on input RNA mass. They recommend 200 mcl beads per 75 mcg of starting RNA, which means that you could theoretically use 2 mcl beads for a 750 ng RNA sample, assuming you could control such a small number of beads in the magnet plate. So this makes Dynabeads cost a little more reasonable. I would be surprised if they were better than Seramag, though.

As for fragmentation, the Illumina TruSeq protocol has a very nice 1-step bead elution/priming/fragmentation that I think is about as convenient as you're going to get. What I don't exactly know is what's in the fragmentation buffer. I'm guessing its Magnesium that you then dilute down for cDNA production, but I'm not sure. Unless you're up for testing it out yourself, this sort of leaves you having to buy the sample prep kit....