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
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
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