Please note that we would need information on the number of samples you are interested in sequencing. Pricing is quoted per sample, with sequencing data output of up to ~5000 cells, without data analysis. Should you require data analysis, please include this in your request. For further information on our data analysis capabilities for single-cell immunogenomic data, please refer to Single-Cell Data Analysis
We generally aim to encapsulate 5000 cells per sample, and the Chromium’s capture efficiency is about 50%. If you provide 10,000 or fewer cells, we will load them all into the Chromium without counting. However, we prefer that you provide at least 50,000 cells so that we can count the cells, check their viability and tailor the input accordingly.
Samples should be fresh cells, or thawed from cryopreserved cells. Samples cannot be fixed with paraformaldehyde/formalin. 10x Genomics has a protocol for methanol fixation, but we have not validated this.
The samples should be single cell suspensions (please use a cell strainer) at 0.7 – 1.2 x 106 cells/mL in PBS + 1% BSA. They should be delivered to SIgN on ice as quickly as possible to reduce RNA degradation. Please refer to the 10x Genomics web site for recommended protocols.
Sort the cells into a 1.5 mL tube containing either medium or PBS + 0.04% BSA (if your cells are more sensitive, up to 1% BSA can be used). We recommend that you stain to exclude dead cells during FACS. Centrifuge the cells, discard the supernatant and resuspend in a volume of PBS + BSA that will yield a concentration of 2 x 106 cells/mL based on the number of cells the FACS machine stated were collected. In our experience, the number of cells recovered after centrifugation may only be 50-60% of this number.
If you do not require V(D)J profiling of either TCRs or BCRs, the 3’ Library will generally give somewhat better sensitivity because reverse transcription begins from the 3’ end of mRNA transcripts. Note that 10x Genomics has discontinued the 3’ v2 Reagent Kit in favour of the v3 chemistry with improved sensitivity. If you require TCR or BCR sequence information, choose the 5’ Library.
The SIgN Immunogenomics platform has the capabilities to do these advanced assays, but we do not offer these on a fee-for-service model. Please contact us to discuss the possibility of working together on a collaborative basis.
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