SORT-seq is a plate-based method of single-cell RNA sequencing. The technique makes use of 384-well microplates, called cell-capture plates, containing well-specific barcoded primers. SORT-seq (Muraro et al. 2016) is a partially robotized version of the CEL-seq2 protocol (Hashimshony et al. 2016).
Cell capture plates offer a flexible platform for single-cell sequencing. Single cells can be sorted into each well of the 384-well plates using a FACS machine or other cell sorting platform. The plates can be stored for longer time periods, and are easy to ship.
Cell capture plates are prepared at our laboratory using high-end dispensing robots. Our experts will fill each well with ten microliters of sterile mineral oil and a tiny nanoliter scale droplet of uniquely barcoded poly-A primers.
The biology behind SORT-seq
SORT-seq is a 3’-based technique, as the primers capture the poly-A tail of mRNAs. Each primer contains a cell-specific barcode and a unique molecular identifier (UMI). It is therefore possible to distinguish well-specific and cell-specific mRNA molecules. The following reactions take place during the processing:
- Cell lysis & primer hybridization
the cells within the wells are lysed by heat, and the primers will hybridize to the poly-A tail of the mRNAs.
- Reverse transcription & second-strand synthesis
These two reactions will generate DNA from RNA. The DNA contains the sequence of the mRNA and the primer.
Since each molecule is now uniquely barcoded, the material of each plate can be pooled. At this point, one plate will become one sample with the material of 384 wells.
- In vitro transcription (IVT)
This reaction will generate many copies of RNA via linear amplification.
- Fragmentation & reverse transcription
The amplified RNA (aRNA) is fragmented into segments of approximately 300-400 base pairs. The reverse transcription reaction will again generate DNA from RNA.
- Polymerase chain reaction (PCR)
The DNA is amplified using PCR. This reaction ensures that there is enough material for sequencing and adds the required sequencing adapters to the material. The resulting cDNA library is ready for Next Generation Sequencing (NGS).
Advantages of SORT-seq
- A low cell input is possible
As SORT-seq is based on 384 well plates, the technique is suitable for as little as hundreds of cells per sample. Plates can be partially filled if cell numbers are very low.
- SORT-seq is flexible
Multiple cell capture plates can be filled will single cells during one or during multiple FACS sort experiments and stored at -80°C for months. The plates can be processed all at once, in small groups, or one by one. It is all possible!
- Specific cell types can be analyzed
The cells are FACS sorted, so a selection of one or more specific cell types can be made.
- FACS parameters can be linked to the sequencing data
When the index files are saved during FACS sorting, FACS parameters can be linked to the sequencing data of specific individual cells.
- Reduced amplification bias
SORT-seq makes use of IVT instead of PCR for the first amplification step. Linear amplification reduces the amplification bias.
When SORT-seq plates are processed, we perform the following reactions:
- Reverse transcription and second-strand synthesis reaction, to generate (unamplified) barcoded DNA in each individual well of each cell capture plate.
- The material from all wells of one plate is subsequently pooled into a single Eppendorf tube.
- In Vitro Transcription reaction (IVT) is performed; a linear amplification step that results in amplified RNA (aRNA).
- Thea RNA is fragmented, and we run it on a Bioanalyzer to check the RNA yield.
- Another reverse transcription reaction is performed, as well as a PCR reaction. These steps result in a cDNA library that contains the right adapters for sequencing. We run another Bioanalyzer to check the quality and concentration of the final cDNA libraries.