The 24th Annual Meeting of the RNA Society will take place from June 11th to June 16th in Krakow, Poland. Lexogen is delighted to welcome you there as a Gold Sponsor, continuing its long-standing support of the RNA Society’s annual meetings and RNA Salons.

Find the Lexogen booth at the exhibition area, attend our poster, join us for a breakfast seminar, and get a chance to win prizes!

Breakfast seminar

Applying Lexogen RNA-Seq technologies to determine transcription & mRNA turnover, RNA-protein interactions, and poly(A)+ & poly(A) RNA 3’ end abundances

Thursday, June 13, 2019

07:45 – 08:45 am

Park Inn by Radisson Krakow

The breakfast will be served at 07:30 am

Chairperson: Lukas Paul (Senior Manager of Scientific Affairs, Lexogen, Vienna, Austria)

While NGS-based RNA sequencing has established itself as a powerful tool for transcriptome analysis, it entails diverse platforms, applications, and protocols. In this seminar, three stories will be presented that reveal how selected technologies from Lexogen – SLAMseq metabolic RNA labeling, the CORALL total RNA-Seq library preparation protocol, and the QuantSeq 3’ RNA-Seq method – can be applied to distinct research questions.

Anica-Scholz_Seminar

Translational regulation of IL32 in the inflammatory tumor microenvironment

Anica Scholz

Goethe-University Frankfurt, Germany

07:45 – 08:05 am

Anica Scholz1, Nicola Boeffinger1, Denise Aigner1, Bernhard Brüne1, Tobias Schmid1

1 Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Germany

Translation is one of the most energy-demanding cellular processes, and therefore is tightly regulated. Changes in translation are commonly observed during tumor development, contributing to various hallmarks of cancer. Tumors are closely associated with inflammation. Specifically, tumors do not only arise in inflammatory sites, they acquire the ability to evade anti-tumor responses and instead corrupt certain immune cells, such as macrophages, to support tumor growth and progression.

Here, we aimed to further characterize the translational changes in tumor cells within an inflammatory tumor microenvironment. To this end, we stimulated human MCF7 breast cancer cells with interleukin 1β (IL1β) and identified translationally regulated targets via ribosome profiling analyses. Among these, the pro-inflammatory cytokine IL32 emerged as an interesting candidate. Interestingly, while translation regulatory mechanisms are commonly mediated by features within the 5’ untranslated regions (5’UTRs) of transcripts, this was not the case for IL32, despite the strong changes in translation in response to IL1β. Instead, corroborated by massively altered protein occupancies, the 3’UTR of IL32 appeared to confer the regulatory properties.

Manfred-Schmid_Seminar

Simultaneous measurement of transcriptional and post-transcriptional parameters by 3’ end RNA-seq

Manfred Schmid

Aarhus University, Denmark

08:05 – 08:25 am

Manfred Schmid1, Agnieszka Tudek2, Torben Heick Jensen1

1 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

Cellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation. Measurement of these two parameters is usually performed independently, complicating analysis as well as introducing methodological biases and batch effects that hamper direct comparison. We developed a simple approach of concurrent sequencing of S. cerevisiae polyA+ and polyA RNA 3’ ends to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample (Schmid et al., 2018). The transcription data generated correlate well with reported estimates and also reveal local RNA polymerase stalling and termination sites with high precision. Although the method by design uses brief metabolic labeling of newly synthesized RNA with 4-thiouracil, the results demonstrate that transcription estimates can also be gained from unlabeled RNA samples. These findings underscore the potential of the approach, which should be generally applicable to study a range of biological questions. We recently applied the same experimental strategy to analyze nuclear RNA exosome-dependent decay pathways in human cells. Highlights from those efforts will be presented at the end.

Reference:
Schmid M, Tudek A, Jensen TH. (2018) Simultaneous measurement of transcriptional and post-transcriptional parameters by 3’ end RNA-Seq. Cell Reports 24: 2468-2478.

Tobias-Schmid_Seminar

Characterization of mRNA dynamics under hypoxia using SLAMseq

Tobias Schmid

Goethe-University Frankfurt, Germany

08:25 – 08:45 am

Rebekka Bauer1, Dominik C Fuhrmann1, Tobias Schmid1, Bernhard Brüne1

1 Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Germany

Reduced oxygen availability, i.e. hypoxia, is an important feature of numerous pathophysiological conditions. In addition to short-term hypoxic episodes, as observed e.g. in the case of ischemic stroke, various disease states, including tumors and inflammatory diseases, are characterized by chronic or intermittent hypoxia. Many cellular responses to low oxygen tensions are mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors consisting of a shared, constitutively expressed β-subunit and one of three α-subunits. Yet, there is increasing evidence that post-transcriptional mechanisms play an important role in the adaptation to hypoxia as well.

In the present project, we aimed to determine the dynamics of mRNA expression changes under acute and chronic hypoxia. To assess the impact of both transcriptional and post-transcriptional (i.e. mRNA stability) regulatory mechanisms in myeloid cells, we employed the recently developed SLAM-seq (thiol-linked alkylation for the metabolic sequencing of RNA) method (Herzog et al., 2017). Specifically, we labelled monocytic THP1 cells with 4-thiouridine (4SU) under acute and chronic hypoxia and analyzed changes in de novo synthesis as well as in the stability of mRNAs in a transcriptome-wide manner.

Using this approach, we were able to assign altered mRNA expression levels in response to hypoxia to changes in de novo transcription, in mRNA stability, or a combination of both. Importantly, our findings shed further light on the exact molecular mechanisms facilitating the adaptation to hypoxia.

Reference:
Herzog VA, Reichholf B, Neumann T, Rescheneder P, Bhat P, Burkard TR, Wlotzka W, von Haeseler A, Zuber J, Ameres SL. (2017) Thiol-linked alkylation of RNA to assess expression dynamics. Nature Methods 14: 1198-1204.

Location

The seminar will take place in Park Inn by Radisson Krakow, a one-minute walk from the ICE Krakow Congress Centre.

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Lexogen poster

Pamela_Moll_Lexogen

Poster #252
Presented during the Wednesday poster session at 20:30 – 23:00.

Cap-dependent linker ligation increases specificity for full-length products compared to template switch reaction

Pamela Moll

Lexogen GmbH, Vienna, Austria

Pamela Moll1, Musashi Tsujita1, Florian Kabinger1, Tomas Dozd1, Michael Ante1, Andreas Tuerk1, Torsten Reda1, and Alexander Seitz1

1 Lexogen GmbH, Campus Vienna Biocenter 5, 1030 Vienna, Austria

Nanopore enables full-length sequencing of RNA or cDNA. A fast and easy way to obtain full-length cDNA is the commonly used template switch reaction. The reverse transcriptase (RT) adds non-templated nucleotides (preferentially C’s) at the end of a transcript which hybridize to abundant template switch oligos. However, non-templated nucleotides can also be added to fragmented RNAs or premature termination sites of the RT. Artificial Spike-in transcripts are an essential part to monitoring the quality of an RNA-Seq experiment, to control NGS sample preparation, base callers and algorithms by adding a ground truth to the NGS experiment. We used Lexogen SIRV™ set 3 containing 69 Spike-In RNA Variant controls, simulating alternative splicing of 7 SIRV genes plus antisense transcription, plus 92 ERCC Spike in controls (External RNA Controls Consortium Spike-In controls, Thermo Fisher Scientific Inc.). ERCCs are monoexonic but cover a concentration range of 6 orders of magnitude. TeloPrime is a full-length cDNA preparation kit offered by Lexogen. Exceptional 5‘-Cap specificity is achieved with the proprietary CAP dependent linker ligation. We capped SIRV set 3 using the Vaccinia Capping Enzyme and protocol from NEB (M2080). Universal Human Reference RNA (UHRR) was spiked in with the capped SIRV set 3 before the sample was subject to a controlled degradation. Nanopore sequencing libraries were made either by using the template switching protocol or a modified TeloPrime protocol (v3) from intact and degraded RNA aliquots. The new TeloPrime v3 Nanopore libraries contain a 12 nt unique molecular Index (UMI) that is introduced with the RT primer, enabling to account for sequencer and PCR errors in high coverage NGS data. Degraded RNA resulted in shorter libraries for the less Cap-sensitive template switching protocol, but little delay in the PCR cycles, while for TeloPrime v3 degraded RNA libraries resulted in significantly less amplifiable library. The analyses of apparent transcript start site distributions by Nanopore sequencing showed a higher cap specificity for TeloPrime v3 than for the template switching protocol. Hence, TeloPrime v3 enables an increased accuracy for transcript 5’ end detection.

Keywords: Cap Specificity, Full-length cDNA, nanopore sequencing

Win a prize!

Lamzac_Lexogen
Lexogen_I_Love_RNA_Stickers

Resolve the Lexogen puzzle and get a chance to win one of 5 air loungers.

Post Lexogen “I Love RNA” sticker on Twitter or Instagram, use the hashtag #IloveRNA, tag @Lexogen, and get a cool RNA T-shirt at our booth.

You can find the puzzle and the sticker in the attendee’s bag or get them at the Lexogen booth.

Looking forward to meeting you soon.

Your Lexogen RNA 2019 team!