[lahoman]

I did not perform Fragmentation for my 1st FFPE sample. Now I am thinking I may need do to do that to narrow the insert size ...

[JakobHedegaard]

Jakob, I have been involved in a piece of work trying to do the same thing - but we just got massive adapter contamination in the final data. What size range was your FFPE RNA peak after RiboZero GOLD but before the ScriptSeq? Ours was between 25-500bp approximately, peak generally around 100.

Regards,
Hedley

RNA size range as yours - but with a sligthly higher peak ~200 nt. How do you purify the library after amplification? Using MiniElute will give you some "primer-dimer" contamination. We are using AMPure XP but you may need to play around with the bead ratio to get rid of the "primer-dimer" without removing too much of the library. Have you considered gel purification?
/Jakob

[HedleyC]

RNA size range as yours - but with a sligthly higher peak ~200 nt. How do you purify the library after amplification? Using MiniElute will give you some "primer-dimer" contamination. We are using AMPure XP but you may need to play around with the bead ratio to get rid of the "primer-dimer" without removing too much of the library. Have you considered gel purification?
/Jakob

Hi Jakob,
The ScriptSeq and later steps were performed by a 3rd party. They used AMPure XP, but it's unclear if they used the right bead ratio to minimise carry-through of the adapter dimers (I suspect not). We are doing some repeat work and also looking at another FFPE kit, so if I find out anything useful, will post it here at some point.
/Hedley

[woodydon]

Dear all,

If you use RiboZero, will non-polyA RNAs dominate the RNA-Seq library? My friend told me that non-polyA RNAs are highly abundant. Will RiboZero end up dilute the polyA RNAs, which should be more important most of the time?

Bests,
Woody

[JakobHedegaard]

@ServiceXS, we obtained enough (>500 ng total RNA) purifiying from 2-3 sections from a FPFE block. For Ribo-Zero we started with 500 ng total RNA but have prepared libraries with 100 ng RNA - but with more variation in quality and yield.
@woodydon, using Ribo-Zero will result in a much more complex library than using poly-dA selected RNA. Depending on the sample conditions (tissue, stage, etc.) some transcripts can dominate the profile - and this applies to both poly-dA and Ribo-Zero treated RNA. Analysing data from Ribo-Zero treated RNA, I think the major challenge is the profiling of both mature and pre-spliced transcripts which introduce some kind of bias.
Cheers, /Jakob

[JakobHedegaard]

@ServiceXS, we obtained enough (>500 ng total RNA) purifiying from 2-3 sections from a FPFE block. For Ribo-Zero we started with 500 ng total RNA but have prepared libraries with 100 ng RNA - but with more variation in quality and yield.
@woodydon, using Ribo-Zero will result in a much more complex library than using poly-dA selected RNA. Depending on the sample conditions (tissue, stage, etc.) some transcripts can dominate the profile - and this applies to both poly-dA and Ribo-Zero treated RNA. Analysing data from Ribo-Zero treated RNA, I think the major challenge is the profiling of both mature and pre-spliced transcripts which introduce some kind of bias.
Cheers, /Jakob