Stingray mRNA Fragmentation NEBNext

[JDSwenson]

Hi all,

I am using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina following mRNA isolation using dT beads.

I've been having some trouble with my mRNA fragmentation time. In this protocol, the fragmentation occurs while eluting the mRNA from the beads into a first strand synthesis buffer/random primer mix. Based on their data using universal reference human RNA, NEB suggests incubating at 94C for 15 min to generate 200 bp inserts, but I am getting 100-150 bp constantly.

Does anybody have experience using this kit and do you have any suggestions regarding how to go about optimizing the fragmentation time to generate 200 bp inserts? Next, I'm planning to fragment at 5, 7, 10, and 12 minutes to see what size my fragments are, but every time I test this it costs time and money. I'd greatly appreciate any input.

Thanks for your time,
John

[nucacidhunter]

Less wasteful approach is contacting tech support but if you want to optimise it yourself then set up one reaction and take aside 2ul samples for running on Bioanalyzer at 8, 10 and 12 minutes. I also wonder if 100-150 bp is insert size after library prep or just after fragmentation. Salts in fragmentation buffer can speed up fragments migration giving a false shorter than actual size. Also fragment size will depend on input and 15 min is for input RNA with high RIN.

[MU Core]

Our experience with the Illumina TruSeq kit suggests a fragmentation of 3-5 minutes is going to be necessary. We achieve an average insert size of 175-200 bp at 3 minutes.

[JDSwenson]

Thank-you for the replies!

I've called tech support and I ran a time series, fragmenting at 5, 7, 10, and 12 minutes. Then I did a cleanup with 2.2x AmPure beads following the RNAClean XP protocol. Attached are my results from the fragmentation time series -- three of the four fragmentations resulted in two distinct peaks, but the fourth looks totally different. Has anybody seen these sorts of sharp peaks after a fragmentation before?

The time for which the mRNA was fragmented at 94 degrees C is in the sample name (e.g. JS16_5 is 5 minutes, JS16_12 is 12 minutes). Everything else was kept the same, including the source of the input RNA.

Parting thoughts ...
I did my original RNA extraction with Trizol followed by a column-based purification and I've read that Trizol often preserves small RNAs, which would explain the ~200 bp peak. My mRNA isolation used oligo d(T) beads, so there shouldn't be any 5s RNAs in the sample (right?) ... but that still doesn't explain why three of the four fragmentations produced peaks instead of curves nor why the length of the fragments seems to have no correlation to fragmentation time ... Any ideas?

Any input whatsoever would be appreciated. I'm pretty baffled.

Thanks a lot

[nucacidhunter]

I have not checked RNA after fragmentation so no idea how it should look like. If you run input RNA on Chip as well that may give some idea about origin of those peaks. cDNA synthesis will prime randomly so the final library fragment insert size will be shorter than fragmented RNA.