Protocol to measure centromeric array size changes using droplet digital PCR-based quantification of higher-order repeats

Centromere length changes occurring during somatic cell divisions can be estimated by quantifying the copy numbers (CNs) of higher-order repeats (HORs), which are nested repeats of monomers that comprise centromeric arrays. Here, we present a protocol for single-cell isolation for clonal evolution followed by droplet digital PCR-based quantification. The assay measures HOR CNs across subclones to determine the frequency and degree of changes in HOR CNs. This protocol tests the underlying molecular mechanisms responsible for rapid centromere sequence evolution. For complete details on the use and execution of this protocol, please refer to Showman et al.1 [Display omitted] •Instructions for isolating single cells for clonal evolution•Procedures for the quantification and preparation of ddPCR samples•Steps for ddPCR technique to measure centromeric array size changes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Centromere length changes occurring during somatic cell divisions can be estimated by quantifying the copy numbers (CNs) of higher-order repeats (HORs), which are nested repeats of monomers that comprise centromeric arrays. Here, we present a protocol for single-cell isolation for clonal evolution followed by droplet digital PCR-based quantification. The assay measures HOR CNs across subclones to determine the frequency and degree of changes in HOR CNs. This protocol tests the underlying molecular mechanisms responsible for rapid centromere sequence evolution.