Single cell sequencing
Single cell sequencing
About single cell sequencing
A typical human cell consists of about 6 billion base pairs of DNA and 600 million bases of mRNA. With such huge amount of sequence, it is expensive and time-consuming to sequence by traditional Sanger sequencing. By using deep sequencing of DNA and RNA from single cell, cellular functions can be investigated extensively. Like typical NGS experiments, the protocols of a single cell sequencing generally contain the following steps: isolation of single cell, nucleic acids extraction and amplification, sequencing library preparation, sequencing and bioinformatic data analysis. It is more challenging to perform single cell sequencing in comparison with sequencing from cells in bulk. The minimal amount of starting materials from a single cell make degradation, sample loss and contamination exert pronounced effects on quality of sequencing data. In addition, due to the picogram level of the amount of nucleic acids used, heavy amplification is often needed during sample preparation of single cell sequencing, resulting in the uneven coverage, noise and inaccurate quantification of sequencing data.
The Single Cell Facility
The Single Cell Facility at the Hubrecht Institute was founded in July 2016, as a response to a growing demand for single cell RNA sequencing projects. The facility enables labs within and outside of the Hubrecht Institute that lack the know-how and equipment to support their research with single cell transcriptome data.
The Single Cell Sequencing Facility uses the SORTseq pipeline. In this pipeline, single cells are sorted into 384 well plates that contain barcoded CELseq2 primers. These plates are provided by the facility. The FACS sort has to be arranged by the client. Besides the plates, preferably some bulk samples (for example 500-1000 cells) are sorted for a RNA-quality check. The plates and bulk samples are returned to the facility, where the plates are processed using the CELseq2 protocol (Hashimshony et al., 2016). After evaluation, resulting DNA libraries are sent out for sequencing to the USF (www.Useq.nl). Once the sequencing data is returned, the sequenced reads are mapped back against the reference genome, and the resulting data is then ready for analysis.
The processing and sequencing take quite some time, and it is expected that after returning the plates to the facility, it will take at least 2 months until the data returns. Every 384 plate costs approximately €1000 (excluding VAT). Sequencing costs and reagents are included in this price.
Whenever you want to make use of the facility, please contact Judith Vivié via j.vivie[at]hubrecht.eu to make an appointment.
Judith in her lab…