Hello,
I'm hoping to get some advice on using the TruSeq RNA kit to produce libraries with 300-350bp insert sizes. Has anyone here found a reliable way to do this with minimal modifications to the TruSeq protocol?
In Appendix A of the protocol, Illumina provides some alternate fragmentation protocols. The most straightforward seems to be decreasing the library insert fragmentation time (the time at 94 degrees). On another post on this forum, a user provided bioanalyzer traces of final library products using 0, 4, and 8 minute fragmentation times (link: http://seqanswers.com/forums/showthread.php?t=13744). They were somewhat surprised to find that at a 0 minute fragmentation time, their final library product demonstrated fragment sizes of over 1kb (compared the Illumina's predicted 130-350bp fragment size). I spoke with Illumina support today in hopes of getting some more insight about this. The person I spoke with said that because of random hexamer priming, the fragments should be relatively small regardless of the size of the fragmented mRNA; she also suggested that perhaps the long RNA product was somehow contaminating the bioanalyzer trace. I don't know enough about bioanalyzer DNA chips to know if that's a possibility. She also said it was very important to let the sample incubate at 4 degrees for a couple of minutes even when using a 0 minute fragmentation time at 94 degrees, and I was unsure if this was the case in the post I linked to earlier.
One of the things I have considered trying is using a 20 second insert fragmentation time. Illumina unfortunately has no data to share about using fractions of a minute in this stage of the protocol. Another possibility I suppose would be to elute the mRNA before fragmentation, reverse transcribe with poly-A primers, and then fragment using our Bioruptor. I'd prefer to avoid that if possible, simply because we have never used it to shear cDNA and we would also have to optimize the bioruptor for the concentrations and quantities produced by the TruSeq protocol.
Any suggestions would be appreciated! Thanks!
I'm hoping to get some advice on using the TruSeq RNA kit to produce libraries with 300-350bp insert sizes. Has anyone here found a reliable way to do this with minimal modifications to the TruSeq protocol?
In Appendix A of the protocol, Illumina provides some alternate fragmentation protocols. The most straightforward seems to be decreasing the library insert fragmentation time (the time at 94 degrees). On another post on this forum, a user provided bioanalyzer traces of final library products using 0, 4, and 8 minute fragmentation times (link: http://seqanswers.com/forums/showthread.php?t=13744). They were somewhat surprised to find that at a 0 minute fragmentation time, their final library product demonstrated fragment sizes of over 1kb (compared the Illumina's predicted 130-350bp fragment size). I spoke with Illumina support today in hopes of getting some more insight about this. The person I spoke with said that because of random hexamer priming, the fragments should be relatively small regardless of the size of the fragmented mRNA; she also suggested that perhaps the long RNA product was somehow contaminating the bioanalyzer trace. I don't know enough about bioanalyzer DNA chips to know if that's a possibility. She also said it was very important to let the sample incubate at 4 degrees for a couple of minutes even when using a 0 minute fragmentation time at 94 degrees, and I was unsure if this was the case in the post I linked to earlier.
One of the things I have considered trying is using a 20 second insert fragmentation time. Illumina unfortunately has no data to share about using fractions of a minute in this stage of the protocol. Another possibility I suppose would be to elute the mRNA before fragmentation, reverse transcribe with poly-A primers, and then fragment using our Bioruptor. I'd prefer to avoid that if possible, simply because we have never used it to shear cDNA and we would also have to optimize the bioruptor for the concentrations and quantities produced by the TruSeq protocol.
Any suggestions would be appreciated! Thanks!
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