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Reposted by Felix Pförtner

Enard/Hellmann Lab @enardhellmannwg.bsky.social · 15/10/2025

Hellnardos at 13th GSCN conference @gscnoffice.bsky.social. We are excited about the next 3 days of stem cell science! 🧫

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Reposted by Felix Pförtner

Enard/Hellmann Lab @enardhellmannwg.bsky.social · 26/08/2025

🎉Excited to share our new preprint: “Improving RNA-seq protocols” We introduce a systematic approach to maximize usable reads in RNA-seq. With this, we created prime-seq2, improving one of the most cost-efficient bulk RNAseq protocols. 👉Check it out: doi.org/10.1101/2025...

doi.org

Improving RNA-seq protocols

Bulk and single-cell RNA-seq are powerful tools for transcriptomic analysis, providing insights into many aspects of molecular and cellular phenotypes. Costs constrain the amount of biological insight...

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

Huge thanks to Eva (shared first author), and to Wolfgang & Daniel, who supervised the project 🙏 🧵9/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

What became very clear throughout the project is how complex and unpredictable a, on the face of it, simple protocol can be. I think we mostly underestimate the complexity of molecular protocols. Also: there’s room for discussion and research into what we consider “usable” reads (discussion).🧵8/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

We think our “funnel strategy” of maximizing usable reads could also be applied more broadly — as a QC metric and as a systematic way to optimize other bulk and single-cell RNA-seq protocols.🧵7/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

We combined these changes into prime-seq2. Across samples and libraries, it delivers +60% usable reads compared to prime-seq. If you’d like to try it: we published a detailed, step-by-step protocol 🧪 👉 dx.doi.org/10.17504/pro... 🧵6/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

For prime-seq, the main loss points were: • total sequencing reads • index assignment • intergenic reads To improve this, we tested many alterations and ended up with: ✅ optimized DNA digest ✅ introduced PTO bonds into PCR primers ✅ redesigned ligation adapters & library PCR primers🧵5/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

This process can be nicely visualized as a funnel: at each step, reads are lost. The funnel makes it clear which stages are most responsible for read loss — and therefore where improvements could make the biggest impact.🧵4/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

We think what matters is the number of reads that can be used for analysis, "usable reads", while controlling for traditional QC metrics. The usable reads number depends on: - valid reads from the flow cell - correct index - filtering - correct barcode - exonic/intronic assignment - unique UMI🧵3/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

Prime-seq has been used extensively in our lab. Over time, several imperfections became clear. During my Master’s thesis, I started on testing many potential improvements. To decide which alterations to keep, we needed a clear QC metric.🧵2/9

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Felix Pförtner @felixpfoertner.bsky.social · 26/08/2025

I am thrilled to finally share our new preprint on prime-seq2 🚀 We improved one of the most cost-efficient bulk RNAseq protocols out there to end up with +60% usable reads. Check it out: doi.org/10.1101/2025... a🧵1/9

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Enard/Hellmann Lab @enardhellmannwg.bsky.social · 07/07/2025

This is the beginning of our office introduction series (1/5): Meet the office of Leo, Antonia with Wilma and Felix Leo 🧠 maps FOXP2 brains Antonia 🧫 reprograms cross-species cells Felix ⚙️ drives Part 2 of every project Wilma 🐶 heads cable-integrity testing by chewing #LabLife #BlueskyScience

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Reposted by Felix Pförtner

Enard/Hellmann Lab @enardhellmannwg.bsky.social · 24/01/2025

The Enard/Hellmann group is now on bluesky 🦋 We are looking forward to reconnecting with everyone here and to welcoming new followers. Let’s keep up great science interactions online!

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