Profile up to 100,000 cells across up to 48 distinct cell samples. After fixation, cells or nuclei undergo four rounds of combinatorial barcoding. Cells are first distributed in 48 wells where an in-cell reverse transcription generates cDNA while appending a well-specific barcode. Two subsequent rounds of pooling and splitting into 96 well plates append two additional barcodes to the cDNA through in-cell ligation reactions. A final split of cells into eight distinct sublibraries is followed by cell lysis. Molecules from lysed cells undergo PCR amplification where a sublibrary-specific fourth barcode is appended to molecules. Together, this yields a total of 48x96x96x8 possible barcode combinations, enough to uniquely profile up to 100,000 single cells. The Split Bio Whole Transcriptome kits are a dramatic improvement on the original SPLiT-seq publication in terms of sensitivity of gene detection.
Split Bio’s fixation allows you to store your single-cell or single-nucleus suspensions for up to 6 months. If you collect your biological samples at inconvenient times of the day or want to barcode cells from samples collected at different time points, the ability to separate sample extraction from downstream cell barcoding could lead to significant time and cost savings. You can multiplex up to 48 of these fixed samples in parallel once you begin cell barcoding. At the end of cell barcoding, you will divide your cells into 8 sublibraries. You can place anywhere from 100 to 12,500 cells per sublibrary (8 sublibraries x 12,500 cells = 100,000 cells). Sublibraries can be processed and sequenced independently from one another, providing you the freedom to fractionate your data and adjust sequencing depth as you choose.
Traditional single cell RNA sequencing methods require complex equipment to first partition individual cells before barcoding their RNA molecules in a cell-specific manner. Our Single Cell Whole Transcriptome kit requires only basic laboratory tools. By using cells themselves as compartments, we can employ a series of split-pooling steps to label all RNA molecules in a given cell with a unique combination of barcodes. This makes it possible for you to perform our standalone kits on your time schedule without the need for upfront costs and instrumentation maintenance fees.