Resource-efficient Strand-seq library preparation in microliter volumes: a one-pot-style adaptation for standard laboratory infrastructure.

Strand-seq is a single-cell sequencing method that preserves directionality of DNA template strands, enabling chromosome-length haplotyping, structural variant discovery, and sister chromatid exchange mapping. The conventional, microliter-scale protocol is reliable but consumable-intensive, requiring five bead-based DNA cleanups and individual per-cell processing across a 96-well plate. A nanoliter-scale, one-pot (OP) adaptation nearly eliminates bead cleanups and plastic consumables in sub-microliter volumes on bespoke nanoarrays, but requires an acoustic dispenser, custom array hardware, and humidity-controlled dispensing not widely available. Here we describe OP-style Strand-seq, a microliter-volume adaptation retaining the central efficiency principles of the one-pot method (pooled MNase digestion, cumulative reagent addition with minimal intermediate purification, and protease-based enzyme inactivation in place of most bead cleanups) while compatible with standard 96- or 384-well plates, liquid-handling robots, and manual pipetting. Relative to the conventional protocol, OP-style Strand-seq reduces tip consumption ∼60% and eliminates three of five cleanup steps, without specialized nanodispensing hardware. • Retains pooled MNase digestion and protease-based enzyme inactivation from the one-pot method, in microliter rather than nanoliter volumes. • Requires two bead-based cleanups instead of five, and no specialized nanoliter dispensing hardware. • Compatible with manual pipetting, liquid-handling robots, or acoustic dispensing in 384-well format, adoptable across diverse laboratory infrastructure.