Grieb, AnissaAnissaGriebOggerin De Orube, MonikeMonikeOggerin De OrubeLee, JaneyJaneyLeeGoudeau, DanielleDanielleGoudeauMalmstrom, RexRexMalmstromWoyke, TanjaTanjaWoykeFuchs, BernhardBernhardFuchs2025-11-272025-11-272025-11-02Springer 978-3-032-07527-7: 43-66 (2025)https://hdl.handle.net/11420/59070As most microbial diversity on Earth remains uncultivated, metagenomics and single-cell genomics have been established as crucial approaches for studying microbial assemblages. While these methodologies provide invaluable insights into the genetic makeup of microbial community members, the genomes recovered most often represent the abundant microorganisms or cells randomly captured. This limits the targeted recovery of genetic material from specific taxa of interest. We here present a comprehensive protocol for the targeted recovery of genomes from uncultivated environmental microorganisms by combining fluorescence in situ hybridization (FISH) with cell sorting. The protocol consists of (i) fixation of cells; (ii) labeling with hybridization chain reaction-FISH; (iii) fluorescence-activated cell sorting (FACS) of labeled populations. The genomic DNA of sorted cells can then be amplified and sequenced using previously established protocols. This procedure can be performed with standard molecular biology and fluorescence imaging equipment, including a FACS machine, and completed within 24 h. This protocol can be adapted for any bacterial or archaeal taxonomic group of interest with available 16S rRNA sequence information.enNatural Sciences and Mathematics::577: EcologyBook Part10.1007/978-3-032-07527-7_4Book Chapter