[cburke11]

If you are talking about in the Truseq protocol, the fragmentation takes place with buffers

[pmiguel]

Result of minimum RNA frag on TruSeq RNA prep

From Illumina's TruSeq™ RNA Sample Preparation Guide, Revision A p43, step 1 of "Incubate RFP":

Place the sealed RBP plate on the pre.programmed thermal cycler. Close the lid and select Elution 2 . Frag . Prime (94°C for 8 minutes, 4°C hold) to elute, fragment, and prime the RNA.

But it is possible to fragment for less than 8 minutes if you want larger amplicon insert sizes.

Typically chemical fragmentation of this sort takes advantage of the catalytic action of divalent cations (eg, Zn++ or Mg++) to accelerate nucleophilic attack of 2' hydroxyl groups on the phosphate backbone, forming 2'-3' cyclic phosphate, and breaking the strand at that nucleotide. However, without heat (94 oC), very little fragmentation will occur.

I brought this up because you may have been like us and wanted longer insert amplicons than the default "150 nt" ones. However Table 12 on page 113 is either misleading or just plain wrong. In it, a 0 minute incubation at 94 oC is said to result in 200 nt inserts.

We tried a 1 second incubation at 94 oC to obtain those 200 nt inserts. However upon running the double stranded cDNA resulting from that RNA with very limited fragmentation time we were shocked to see this size distribution on an Agilent High Sensitivity Chip:



Average size was 1200 bp! That is great for people who prefer to sonicate cDNA for their fragmentation step. But you can see that a very small proportion of the cDNA will be in the ~200 bp size range. So I don't know what Table 12 means. I tend to think it is just wrong.

Anyway, I ask again: how long did you incubate at 94 oC and what method did you use to heat the samples?

--
Phillip

[OnUrMark]

Hi Phillip,

Did you get to the bottom of the fragmentation time and median insert length issue? I spoke to a lab that uses 6 seconds, but the only person in the lab did not know what size fragment resulted.

Thanks for your help!

[NextGenSeq]

Use the NuGEN Ovation RNA-Seq kit. Illumina's kits are among the worst out there.

[OnUrMark]

Thanks for the advice, but I have the TruSeq RNA kit and all necessary consumables in hand. I need to learn how to make this kit work well for us.