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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 ...
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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?Originally posted by HedleyC View Post
/JakobComment
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Hi Jakob,Originally posted by JakobHedegaard View Post
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.
/HedleyComment
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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,
WoodyComment
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@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, /JakobComment
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@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, /JakobComment
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Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM -
With the launch of new single-cell sequencing platforms in 2026, the field stands at an exciting inflection point. This article surveys the most impactful advances in the field and discusses how they’re reshaping research in cancer, immunology, and beyond.
Introduction
Single-cell sequencing technologies have undergone remarkable advances over the past decade, transitioning from low-throughput experimental approaches to highly scalable platforms capable of...05-22-2026, 06:42 AM
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