VASA-seq for full-length single-cell sequencing
VASA-seq* is a single-cell sequencing platform for full-length, total RNA sequencing. Like SORT-seq, VASA-seq is based on 384-well cell capture plates and FACS sorting.
In contrast to other single-cell sequencing technologies, VASA-seq does not rely on primers that bind to the endogenous poly-A tail of mRNA molecules. Therefore, VASA-seq allows us to analyze non-coding RNA species and immature mRNA molecules simultaneously, all at single-cell level.
How it works in brief: Single cells are sorted into the wells of a prepared 384-well cell capture plate. RNA molecules are barcoded with primers that contain a cell-specific barcode. After barcoding, double-stranded cDNA is generated, after which the contents of the wells are pooled and the RNA is amplified with in-vitro transcription (IVT). After IVT, the sample is prepared into an Illumina sequencing library.
*Disclaimer: VASA-seq is a new and yet unpublished technology that we license from the KNAW and Oncode Institute. Until VASA-seq is published, there is an embargo on publishing VASA-seq data and results.
What you can do with VASA-seq
VASA-seq is highly suitable for single nucleus sequencing, since it does not depend on polyadenylation and can capture the immature RNA molecules present in the nucleus that do not have a poly-A tail yet.
Furthermore, you can use VASA-seq to sequence total RNA to study non-coding RNA species such as long non-coding RNAs and rRNAs at single-cell level.
Lastly, because VASA-seq generates full-length information on the RNA molecules, you can study RNA isoforms at single-cell level. As it can be used to separate reads coming from introns or exons along the full length of the RNA molecule, VASA-seq provides the ideal data to infer RNA velocity within your dataset.
Key benefits of VASA-seq
- Analyze RNA molecules at full length
VASA-seq generates full length coverage of the RNA, all the way from the 5' to the 3' end. This means you get information on intron/exon boundaries and SNPs.
- Analyze coding and non-coding RNA species
VASA-seq is able to sequence non-polyadenylated RNAs. This includes immature mRNAs and non-coding RNAs.
- Compatible with all sample types
VASA-seq works with low quantities of cells. As long as you can fill (part of) a 384-well plate by FACS sorting, you can generate single-cell sequencing libraries
How does it work
Step 1: FACS sorting
Begin by ordering our cell capture plates, which we ship worldwide. You then sort your single-cells into the 384-well cell-capture plates and send the plates back to us for processing. If you don't want us to process all the plates at once, just let us know which ones to start with.
Step 2: Plate processing
Once we've received your plates, we'll process them using the SORT-seq protocol. After barcoding the mRNA of your single-cells, we'll pool the material from one plate to generate one sequencing library per plate.
Step 3: Sequencing and data analysis
We'll send the sequencing libraries to one of our partners for sequencing. When we get the data back, we'll perform a preliminary data analysis, then send the data back to you.
As with all of our services, we provide ongoing support from our single-cell sequencing specialists. For each SORT-seq project, we provide the following:
- Regular email updates during plate processing
- Raw sequencing data (FASTQ files)
- A QC report per plate
- Mapped count tables for downstream analysis
- A preliminary clustering analysis, done with the Seurat pipeline.
- A preliminary differential gene expression analysis
Once we have sent you the data, we'll invite you to schedule a call with one of our scientists to go over any additional questions you may have.
That's when we can discuss any additional data analysis you may need.
Our throughput time is 4-6 weeks, from the moment we receive the plates.
All fresh cell types/nuclei that can be FACS sorted (live/dead), are suitable. Methanol-fixed cells that are rehydrated before sorting are also compatible with VASA-seq.
We recommend keeping 8 wells empty as no-template negative control, so it is possible to analyze up to 376 cells per plate.
Yes, both technologies use the same cell capture plates.
You can order the cell capture plates via the plate order form.
Not sure if VASA-seq is right for your project? Explore our other services.
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