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.

You will receive a delivery mail containing:

• a report including experimental design and run conditions
• instructions on how to access your data through our delivery server

CTG uses secure LSENS servers hosted by LUNARC for internal data management and analysis. All data is delivered to the customer via a dedicated secure file server hosted at Lund University.

After sequencing, data will be kept by CTG for 3 months and subsequently deleted. We strongly recommend you to backup, i.e. that have at least two copies. CTG can not help if your data gets corrupt or lost!

The CTG RNA analysis data delivery will include:

• FASTQ files  
  Demultiplexed data files generated from the Illumina sequencing run.
  BAM files     
• Read Mapping: alignment of reads to a specified reference genome (human, mouse or rat).
  Expression count matrix    
• Assembly of the alignments into full transcripts and quantification of the expression levels of each gene/transcript.
  Quality Control (QC) files    
• A number of RNA-Seq quality control analyses summarized in FastQC and MultiQC reports.

For additional study design and data analysis services please contact NBIS – National Bioinformatics Infrastructure Sweden.