{"id":381,"date":"2020-03-10T15:55:18","date_gmt":"2020-03-10T14:55:18","guid":{"rendered":"http:\/\/wp.unil.ch\/gtf\/?page_id=381"},"modified":"2023-04-28T19:36:56","modified_gmt":"2023-04-28T17:36:56","slug":"single-cell-experiments-10x","status":"publish","type":"page","link":"https:\/\/wp.unil.ch\/gtf\/single-cell-experiments-10x\/","title":{"rendered":"Single cell (10X)"},"content":{"rendered":"\n

10X Genomics Next GEM technology<\/span><\/strong><\/p>\n\n\n\n

GTF operates 10X Genomics<\/a> solutions for high throughput single cell profiling.<\/p>\n\n\n\n

We run both the standard Chromium and the newer Chromium X which required for the Single cell fixed RNA profiling<\/a> application from 10X genomics (human and mouse only) and the High Throughput assay (twice more cells processed for roughly the same price).<\/p>\n\n\n\n

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10X Genomics trick is based on single cell\/nucleus emulsion. A DNA barcode, unique in each droplet, is linked to DNA\/cDNA. The emulsion is broken and the subsequent sequencing library generation steps are performed bulk.<\/p>\n\n\n\n

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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. It requires cells are fresh and high viability. It works on any organism, provided the RNA is polyadenylated.<\/p>\n\n\n\n

With the the fixed cells protocol<\/strong>, gene expression is achieved by sequencing RNA bound probes. Fixation with PFA leaves way more flexibility for sample preparation. Probes are however available only for human and mouse transcriptome. In addition, only gene expression applications are possible (not ATAC-Seq or VDJ sequencing).<\/p>\n\n\n\n

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Up to 10\u2019000 cells\/nuclei can be assayed in a single well, i.e. up to 80\u2019000 cells per chip. However, the more cells or nuclei are loaded, the more multiplets may be present (see for instance this 10X Genomics note<\/a>). In addition, more cells mean higher sequencing effort required (see below). A common target is in the range of 8\u2019000 cells\/nuclei per well.<\/p>\n\n\n\n

With the high throughput assay, these numbers are doubled for roughly the same 10X processing cost. By using cell multiplexing (see below), it is possible to recover up to 30’000 cells per well. This is however bound to several limitations (please inquire).<\/p>\n\n\n\n

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Applications<\/span><\/strong><\/p>\n\n\n\n

Main 10X Genomics single cell applications are:<\/p>\n\n\n\n