RNA Sequencing Library Preparation – How Low Can You Go?

Overview

Course Overview

Aim of this course is to prepare RNA-Seq libraries for Illumina sequencing by using the novel NEBNext Ultra II Directional RNA kit for low input applications, not including single cells transcriptomics.

Throughout the course, we will be generating libraries from ultra-low as well as from normal amounts of RNA (1 ng to 50 ng) input to better understand the impact of input amounts and PCR cycles on the bioinformatics dealing with complexity, coverage, duplication rates and uniformity of libraries. The course participants will be guided with tips and tricks through the work flow. Finally, the bioinformatics analysis of the data will be discussed. Selected participants who – PRIOR to the course – will be able to prove their samples have required quality according to provided specifications will have the chance to bring maximum two of their own eukaryotic total RNA samples to process. At least 100ng of sample will be required for attendee samples.

Audience

This course is directed towards wet-lab biologists, scientists and technicians who want to apply transcriptome analysis to “tricky” samples and learn the basis and best practise of library preparation. Knowledge of next generation sequencing technology is desirable but not essential.

Modules/Resources

– Nucleic acid (RNA) processing
– NGS
– Introduction to RNA-Seq data analysis

Learning Outcomes

Preparation of libraries from low starting material and dealing with analysing of the data.

Speakers

Claudio Asencio

EMBL Heidelberg

Germany

Nayara Azevedo

EMBL Heidelberg

Germany

Vladimir Benes

EMBL Heidelberg

Germany

Andreas Burchert

Universitätsklinik Marburg

Germany

Ferris Jung

EMBL Heidelberg

Germany

Keerthana Krishnan

NEB

USA

Jonathan Landry

EMBL Heidelberg

Germany

Dinko Pavlinic

EMBL Heidelberg

Germany

Jan Provaznik

EMBL Heidelberg

Germany

Björn Textor

NEB

Germany

Scientific Organisers

Vladimir Benes

EMBL Heidelberg

Germany

Björn Textor

NEB

Germany

Course Organisers

Maria Mercedes Bacadare Goitia

EMBL Heidelberg

Germany

Programme