After the third round barcoding and before lysis, cells or nuclei will be pooled and subsequently split into distinct populations we call sublibraries. Each sublibrary will contain a different 4th barcode (the Illumina index) and can be sequenced separately. You will count cells or nuclei before splitting them into your sublibraries, and thus you can choose the number in each. In practice it can be useful to have at least one sublibrary with few cells or nuclei; this can provide an estimate of gene detection per cell at deeper sequencing while using fewer sequencing reads.
