RNA Library Preparation and Sequencing

mRNA profiling

• Illumina® Stranded mRNA Prep, Ligation as standard workflow*

*Sequencing is performed on NovaSeq 6000, with read structure of 2 x 150 bp and aiming for 25M reads per sample.

Total RNA profiling

• Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero as standard workflow^
• TaKaRa SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian for low input and poor quality samples.

^Sequencing is performed on NovaSeq 6000, with read structure of 2 x 150 bp and aiming for 50M reads per sample.

If you are interested in collaborating with us on development of new protocols, or if you have a suggestion for the implementation of a novel method, please contact tina [dot] catela_ivkovic [at] med [dot] lu [dot] se (Tina Catela Ivkovic).

General information before starting an RNA project

• RNA samples need to be of high quality, recommended RIN > 8.0
• Freeze and thaw samples as few times as possible and work with the RNA on ice to avoid degradation.
• Measure RNA integrity by capillary electrophoresis method (Fragment Analyzer, Bioanalyzer or TapeStation) and provide RIN values for each sample when requesting a project. This analysis is also available at CTG.
• RNA samples should be of high purity, recommended OD260/280 ~2.0 and OD260/230 ~1.8-2.2 based on spectrophotometric measurement (e.g. by Nanodrop).
• All samples must be diluted/normalized based on concentration measurements performed by a fluorometric method (e.g. Qubit). Measurements based on spectrophotometric method (e.g. Nanodrop) are not acceptable.
• Sequencing depth and read length requirements can vary depending on the goals of the project. If there is a requirement for read length or depth deviating from our standard protocols (see above), please specify when requesting a project in iLab.

Sample input

• RNA input amounts differ between assays. Please see table below for the required sample concentration and delivery volumes. Lower concentrations and volumes can be discussed.
• Always dilute samples in nuclease-free H₂O

Delivery of samples to CTG

RNA should be delivered to CTG aliquoted on dry ice in:

• an 8-tube strip labeled with Sample ID or Sample number (i.e. 1, 2, 3...) on the lid and side of the tube. 
• a fully skirted 0.2 ml plate with cold resistant seal (can be provided by CTG). Samples bust be placed column-wise (A1, B1, C1...)

Requesting a project at CTG

To initiate a project using our available services, please start a project in iLab. You can login using your LucatID (SWAMID) on the Lund University iLab webpage.