Plate-based/split-pool single-cell sequencing

In split-pool barcoding methods, single-cell identity is generated through successive rounds of distributing fixed cells across plate wells, where each round introduces a distinct barcode.

In split-pool barcoding methods, single-cell identity is generated through successive rounds of distributing fixed cells across plate wells, where each round introduces a distinct barcode. After each barcoding step, cells are pooled and redistributed, and the final combination of barcodes uniquely labels the cDNAs from each individual cell.

The strengths of these methods include:

• The usage of fixed cells, allowing for sample batching over time
• High number of cells can be targeted, depending on the kit, with cost efficient analysis of millions of cells
• Extensive multiplexing of samples possible: where sample ID is traceable by the initial round of barcoding/well.
• Several -omics available, depending on the kit, including V(D)J and CRISPR analysis

Split-pool workflows are especially advantageous when analysing large sample or cell numbers. However, these methods, including sample fixation, freezing, thawing and several rounds of barcoding, are associated with a higher degree of sample loss than some other single-cell workflows. As a result, it is necessary to begin with more cells than the final target number.

These approaches are particularly well suited to applications where sufficient starting material is available, and where some sample loss is acceptable. They can also be advantageous when working with complex or heterogeneous samples, where enrichment of specific subpopulations is not feasible or desired.