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

## TL;DR

This paper provides a detailed protocol for recovering and sequencing ssDNA from coacervate droplets, which are synthetic cell models that can trap nucleic acids.

## Contribution

The novelty lies in the sequence-independent library preparation and data analysis pipeline for studying ssDNA in metabolically active coacervates.

## Key findings

- A protocol for time-resolved harvesting and deep sequencing of ssDNA from coacervate droplets is described.
- An analysis pipeline is introduced to assess enrichment dynamics and sequence distributions across different droplet systems.

## Abstract

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

•Protocol to prepare and harvest ssDNA from fueled complex coacervates•Sequence-independent, orientation-specific ssDNA library preparation for NGS•Comprehensive data analysis pipeline to standardize results across research groups

Protocol to prepare and harvest ssDNA from fueled complex coacervates

Sequence-independent, orientation-specific ssDNA library preparation for NGS

Comprehensive data analysis pipeline to standardize results across research groups

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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 NGS, along with an analysis pipeline to assess enrichment dynamics and sequence distributions. The protocol is adaptable to diverse droplet systems.