title: Asteroid-associated bacterial metagenome B7
design: Samples of asymptomatic asteroid tissues (~ 1cm2) were excised from dorsal surfaces and lesioned tissues were excised from symptomatic asteroids, and then homogenized in 10mL of 0.02  m filtered phosphate buffered saline using the NutriBullet for 1 minute. Homogenates were allowed to settle for 5 min before 1 mL supernatant was removed and placed into sterile microcentrifuge tubes. The homogenate (400  L) was purified on PERCOLL step gradients (800  L each step at 70% and 30%) to remove host cells. Gradients were centrifuged at 12,000 x g for 90 min (4oC). The interface between 30% and 70% was determined with reference to a dense E. coli culture run on an identical gradient. The interface was removed and DNA extracted using the ZR Bacterial & Fungal DNA kit (Zymo Research) following manufacturer s recommendations. The interface fraction was then targeted for metagenomic library preparation. DNA was extracted from the cell fraction and amplified using the GenomePLEX kit (Sigma Aldrich)