Uneniched Black Molly
A single ~5 cm Black Molly was euthanized in MS-222, rinsed and homogenized in 30 ml 1X PBS with dissection scissors. The homogenate was vortexed for 10 minutes to disassociate microbial cells from host tissue, and then filtered through a 5 µm Isopore polycarbonate filter (Millipore). The filtrate was spun down at 14,000 xg for 10 minutes to pellet microbial components of the fish microbiome. The pellet was then dried and re-suspended in 600 µl Puregene Yeast/Bac Kit cell lysis solution (Qiagen), and DNA purification was performed as per manufacturer’s protocol.Illumina library construction and sequencing (MiSeq, GAIIx): genomic DNA (500 ng) was sheared for using a Covaris S2 device to obtain an average fragment size of ~350 bp. Illumina paired-end sequencing libraries were constructed using the NEBNext® DNA library prep reagent set for Illumina (NEB #E6040) following the standard Illumina sample preparation protocol. PCR library amplifications were performed with an MJ Research Thermo Cycler PTC-225. Illumina PE 1.0 and 2.0 primers, or PE 1.0 and 2.0-derived indexing primers, were used to amplify adapter-ligated library fragments by PCR. Libraries were amplified using optimized PCR conditions described.