The 5 'Single Cell V(D)J Phenotyping & Gene expression analysis workflow is a 5'-based workflow for parallel analysis of the T cell receptor or B cell receptor repertoire and gene expression in individual cells. Individual cells are packed into tiny water-in-oil droplets using the Chromium Controller along with a gel bead containing a mixture of different barcodes. In this reaction volume of about 1 nl, the cell is lysed and the mRNA molecules are barcoded and transcribed into cDNA. Subsequently, the resulting emulsion is chemically broken and the cDNA amplified in a sequence-independent manner. For the analysis of the TCR or BCR repertoire, a small part of this amplified cDNA is used as template for a TCR or BCR specific target enrichment. The remaining cDNA is usewd as input for an Illumina-compatible library preparation for the analysis of single cell gene expression. After sequencing, the sequences can then be bioinformatically assigned to the individual cells or transcripts on the basis of the attached barcodes.
It can be freely chosen whether within a sample only the TCR or the BCR repertoire should be analysed, or whether these analyses should be coupled with the gene expression. In principle, all combinatorial possibilities from TCR, BCR and gene expression analysis are possible.
A maximum of 8 samples can be processed in parallel on a single chip of the Chromium controller. For each sample, between 500 and 10,000 cells can be analysed. Approximately 60% of the cells used as input are actually analysed. Meaning that If 10,000 cells are to be analysed for a sample, 17,000 cells are needed as starting material.
As input material, a single cell suspension is required. This suspension has to meet two key criteria in order to deliver high-quality data.
On the one hand only truly single cells may be present in the suspension. If cell aggregates are present in the suspension, they have to be removed. On the other hand, the cells should have the highest possible vitality. The suspension should not contain more than about 20% of dead cells. An excessive amount of dead cells leads to a high background signal. If your samples does contain a higher proportion of dead cells it may be necessary to remove the dead cells.
After receiving the samples in our laboratory, a quality control is routinely performed determining both cell count and vitality of your sample.
Since for this type of analysis the input material is cell material, please clarify in advance with us whether your material may be processed in our laboratory!