RNA Library Preparation and Sequencing
Our RNA service includes library preparation for mRNA, total RNA and sequencing on Illumina sequencing platforms. See below for available kits.
Transcription levels for studies of differential expression
- Illumina TruSeq Stranded mRNA kit is our standard kit for library preparation. The messenger RNAs are selected through poly-A enrichment.
- SMART-Seq v4 Ultra Low Input RNA kit (non-Stranded) from Takara combined with Illumina’s Nextera XT Kit can be used when having very small amount of RNA.
Sequencing is mainly performed on NovaSeq, reading 2 x150 bp and aiming for 25M reads per sample. Optimal numbers of a sequencing kit will be 32, 64, 164 or 400 samples
Whole Transcriptome for studies of the entire population of transcribed RNA (coding and noncoding RNA)
- TaKaRa SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is specifically designed for very low input total RNA samples.
Sequencing is mainly performed on NovaSeq, reading 2 x150 bp and aiming for 50M reads per sample.
General information before starting RNA project
Please read through these following steps:
- Your samples need to be of good quality as degraded or low amounts of starting material can yield low complexity libraries, see sample requirements below.
- Freeze and thaw samples as few times as possible and work with the RNA on ice to avoid further degradation.
- Million reads (read depth) and read length can vary depending on the goals of your RNA-sequencing study. Please inquire if you want read length or depth deviating from our standard procedures (above).
- Illumina TruSeq Stranded mRNA kit is kept in stock and you may order any number of samples when using this protocol. However, note that when ordering a project with fewer samples than for an entire NovaSeq sequencing kit there may be a delay in sequencing time since the project is batched together with other projects. Of course, you can always order a whole sequencing kit even if you do not have entire set of samples.
Sample concentration and quality check
Before handing in your samples we recommend you to:
- Define purity of each sample using a spectrophotometrical method (Nanodrop). Ratio: OD260/280 ~2.0 and OD260/230 ~1.8-2.2.
- Verify concentration of samples using a fluorometric assay (Qubit, Quant-it).
- Measure quality using a capillary electrophoresis method (Fragment Analyzer, Bioanalyzer or TapeStation) that can measure RNA integrity by providing RNA integrity Number (RIN) values.
- RNA input amounts differ between assays. Please see table below for required sample concentration and delivery volumes.
- Total RNA amount recommended
- Always dilute samples in nuclease-free H2O
Lower concentrations and volumes can be discussed.
Delivery of samples to CTG
RNA should be delivered aliquoted on dry-ice in
- strip labeled with Sample ID or Sample no. (ie. 1, 2, 3....) on lid and side of tube
- fully skirted 0,2 ml plate with cold resistant seal - can be provided by CTG (place samples column wise, A1, A2…..)
Data Analysis and Delivery
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 month 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!
*Lower concentrations and volumes can be discussed.
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.
Development of protocols
If you are interested in collaborating with us on our protocols under development, or if you have a suggestion for the implementation or development of a novel method, please contact Tina Catela Ivkovic (tina [dot] catela_ivkovic [at] med [dot] lu [dot] se).
To initiate a project using our available services, please start a project in iLab. ILab is a managing software for core facilities and increases the transparency of projects and quotes.
We have several user guides available. A quick guide for PIs to set up a group/lab, a starting guide for further lab members and a more detailed guide providing information on all functions in iLab. If you have any questions regarding iLab please write to CTGiLAB [at] med [dot] lu [dot] se.
You can login using your LucatID on the main Lund University iLab webpage.
You can also use this link, which brings you directly to our Service Request site after login.
Consultation and preliminary quotes
If you want to schedule a meeting to discuss your project or want to get a preliminary quote, choose “Consultation”, fill out the form, save it and click on “send request” at the bottom of the page. We will process your request as soon as possible.
If you are ready to start a project click on one of the available project request options, fill out the Contact & Invoicing Information as well as project specific forms, save them and click “send request” at the bottom of the page. We will check the forms and send you a quote.
Contact for service
CTGservice [at] med [dot] lu [dot] setarget="_self"