With the fixed cells kit, the multiplexing barcode is brought with the RNA detection probe. Multiplexing can also be achieved with DNA barcoded antibodies as a process called Cell Hashing So far, it is supported with the 3′-gene expression kits only. This saves reagents in case many samples have to be run in parallel, but decrease the number of individual cells assayed for each condition.ġ0X Genomics proposes an official solution with the CellPlex technology. The process is based on an extra DNA label added to the cells (or nuclei), or on barcoded probes when working with fixed cells (see below). Samples may be multiplexed before being loading on the 10X Chromium chip. Given there is also generally around 50% yield for the cell preparation step (cell sorting and subsequent washings), we recommend starting the preparation procedure with at least 4-fold the amount of targeted cells/nuclei.Īs a starting point, please refer to the following 10X genomics documents: Sample Preparation Tips for Single Cell Gene Expression (transcriptomics applications), Nuclei Isolation for Single Cell ATAC Sequencing (ATAC-Seq application), or Nuclei Isolation for Multiome (Gene Expression + ATAC seq from the same nuclei). This means that only ≃ 50% of the cells/nuclei loaded in the chip’s wells will actually be assayed. The Chromium loading efficiency is in the range of 40% to 60%. In any case, we strongly recommend that pilot experiments are performed in order to validate the cell preparation procedures. Data quality can only be assessed after sequencing, meaning that expensive 10X and Illumina reagents are engaged, whatever the outcome is. We also operate the Luna-FX7 which can take advantage of AO/PI staining to accurately distinguish cells from debris.įailure to provide good quality starting material will inevitably result in suboptimal, sometimes useless, results. You may want take advantage of our Moxi V cell counter which is optimal for several 10X Genomics applications (see this technote). It is more than highly recommended that a final quality control step is performed on the very final material you provide us ( disclaimer). In particular, cell count and viability output metrics from cell sorters cannot be taken for granted. concentration by centrifugation) or waiting time can impact cells/nuclei quantity and quality. Nuclei preparation is even more challenging since viabilitycheck does not apply (see 10X note).Īny manipulation (e.g. Cells/Nuclei must be perfectly dissociated, their density accurately quantified, and viability must be above 70% (for transcriptomics applications). The cells/nuclei suspension quality is crucial for a successful single cell project. This is however bound to several limitations (please inquire).Ĭells or nuclei preparation is of user’s responsibility ! By using cell multiplexing (see below), it is possible to recover up to 30’000 cells per well. With the high throughput assay, these numbers are doubled for roughly the same 10X processing cost. A common target is in the range of 8’000 cells/nuclei per well. In addition, more cells mean higher sequencing effort required (see below). However, the more cells or nuclei are loaded, the more multiplets may be present (see for instance this 10X Genomics note). Up to 10’000 cells/nuclei can be assayed in a single well, i.e. In addition, only gene expression applications are possible (not ATAC-Seq or VDJ sequencing). Probes are however available only for human and mouse transcriptome. Fixation with PFA leaves way more flexibility for sample preparation. With the the fixed cells protocol, gene expression is achieved by sequencing RNA bound probes. It works on any organism, provided the RNA is polyadenylated. It requires cells are fresh and high viability. The standard gene expression assay is based on the generation of full length cDNA generation (primed at polyA tail) followed double strand cDNA amplification and generation of a 5′-end or 3′-end sequencing library generation. The emulsion is broken and the subsequent sequencing library generation steps are performed bulk. A DNA barcode, unique in each droplet, is linked to DNA/cDNA. 10X Genomics trick is based on single cell/nucleus emulsion.
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