This protocol is aimed at amplifying the 18S rRNA hypervariable region 9 (18S V9) in eukaryotes with a focus on microbial eukaryotes. Amplicons generated using this protocol can then be sequenced using the Illumina platform. The primers (1391F, EukBr) utilized in this protocol are based on the primer utilized in Amaral-Zettler et al 2009 and the Earth Microbiome Project (EMP). This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASAvdgdg), and the U.S. Integrated Ocean Observing System (IOOS) Program Office. CitationsAmaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM (2009) A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes. PLoS ONE 4(7): e6372. doi:10.1371/journal.pone.0006372Earth Microbiome Project - http://www.earthmicrobiome.org/emp-standard-protocols/18s/