Lcm pen membrane slide

A small cube (~0.5 cm³) of RNALater preserved sample was washed in sterile water three times to reduce salt crystals. The tissue was then crushed gently on a 70 μm filter which was then washed with sterile water (500 μL). The filtrate was centrifuged at 500 × g for 5 min at 4 °C to pellet larger sponge cells and particles. The resulting pellet was then suspended in water (100 μL) and a 10% dilution (50 μL) was spread on an LCM PEN membrane slide (ThermoFisher Scientific, USA) and allowed to air dry. Chemobacteriocytes were identified as distinct round particles with diameter of 15–25 μm and isolated by laser microdissection on an MMI Cell Cut LCM system (mmi). 100 bacteriocytes were collected and 50 background membrane cuts were further collected as a negative control. DNA was extracted from both the cells and negative control using the Masterpure complete DNA extraction kit following manufacturer’s protocol (Epicenter, Madison, WI, USA) and used for Illumina library preparation and metagenomic sequencing as described above (Supplementary Table 1). BLASTn (e-value cutoff of 1 × 10⁻²⁰) was used to assign metagenomic reads to the Ca. E. renieramycinifaciens chromosome, p-ren, and sponge mitochondrion.