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- How many samples should I run? The number of samples should be determined based on your experimental goals. We can support up to 8 samples each day, however it is highly recommended that the investigator perform a pilot experiment before committing to large scale experiment.
- How many cells do I need to provide and how many cells can I expect to get information for? We recommend loading between 10k live cells (for non-hashing experiments) and 20-30k live cells (for cell hashing experiments). Therefore, please provide at least double this cell number, so that there are enough cells to re-load the sample if a wetting failure occurs during the 10x encapsulation. The capture rate of 10x is approximately 60%, depending on cell type and cell quality.
- How should the cells be prepared for 10X? It is recommended that investigators should optimize their cell isolation procedure prior to 10X experiments. In general, we recommend cell viability of >85% for optimal cell input. The Human Immunology Center Flow Cytometry Sorting Core can be used to isolate viable cells or there are protocols available for cell isolation by MACs beads. For single cell RNA-seq, we ask you to resuspend your cells in 0.4%BSA/PBS. We can provide this buffer, or it is fine to prepare it yourself using nuclease free, filtered reagents. For single nuclei RNA-seq, 1% BSA/PBS with 0.2U/μl RNase Inhibitor is required (we can provide the 1% BSA/PBS, but you will need to supply your own RNase Inhibitor). For ATAC-seq, we can provide the 20X Nuclei Buffer – please dilute 1:20 in nuclease-free water before use.
- How many reads per cell can I expect? We aim to provide at least 30k reads/cell for gene expression libraries, as recommended by 10x Genomics. For Cite-seq libraries, we provide a sequencing depth of 5,000 reads/cell. If you are using more than 100 TotalSeq™ antibodies, we will increase the read number to 10,000 reads/cell. For cell hashing libraries, we provide a sequencing depth of 5,000 reads/cell.
- What are the advantages and disadvantages of cell hashing technology? Using cell hashing technology, samples treated in different conditions can be pooled together, thereby decreasing batch effect. Also, it is cost-effective when pooling several samples. However, the main disadvantage of this technology is that fewer cells from the individual conditions can be loaded. For example, when loading 30k total cells in a cell hashing experiment, if you plan to pool 6 conditions in one 10x run there will be 5000 cells/condition, rather than the 10k cells we would normally load in a non-hashtag labelled sample. In addition, the doublet rate with 30k cell input will be much higher than 10k cells, so the number of cells passing QC will be less.
- How do I know if my cell type is compatible with cell hashing technology? To perform cell hashing, you need to make sure:
- Your cells can be labelled with hashtag antibodies. For human samples, the hashtags are made of two antibodies that recognize ubiquitous surface markers, CD298 and β2 microglobulin, each conjugated to the same oligonucleotide containing the barcode sequence. For mouse samples, the surface markers are CD45 and H-2 MHC class I. The conjugates are already pre-mixed and ready to use.
- Your cells can maintain a high viability after the labelling process. We can provide the protocol and relevant staining buffers to researchers to perform a ‘mock’ labelling experiment and obtain a final viability assessment. We require the viability of the submitted samples to be no less than 85%.
- Does the core provide cell hashing antibodies? Yes, we can provide the relevant cell hashtag antibodies, Fc blocking reagent, and staining buffer. After you book your experiment, please coordinate with us to pick up those reagents one day in advance.
- Does the core provide Cite-seq antibodies? No, the core does not provide Cite-seq antibodies. Please order the antibody panels from Biolegend yourself. Please confirm with us the Total-seq format you are planning to use (e.g. Totalseq A or B or C)
- Do I need to pool the cells from different populations myself if I plan to do Cell hashing? Yes. You will need to count the cell number of each population, pool to the required proportions (usually in equal numbers) prior to submitting the pooled sample to us. We will then perform a confirmatory assessment of the cell concentration and viability of the sample prior to running on the 10x Controller. However, if you are using our on-site FACSorting Facility (the HIC Flow Core) the individual post-sorted hash-tagged cell populations can be counted and pooled, per the client’s instruction, by us, if preferred.