Protocol for the recovery and deep sequencing of short ssDNA pools from transient, fuel-dependent coacervate droplets

Complex coacervate droplets are synthetic cell models that sequester nucleic acids. Here, we present a protocol for the recovery and deep sequencing of single-stranded DNA (ssDNA) from metabolically active coacervate droplets. We describe time-resolved harvesting, ssDNA recovery, and sequence-independent library preparation for next generation sequencing (NGS), along with an analysis pipeline to assess enrichment dynamics and sequence distributions. The protocol is adaptable to diverse droplet systems. For complete details on the use and execution of this protocol, please refer to Machatzke et al.¹.