RNA-seq of bacteria in Leptogorgia alba: sea octocoral
Leptogorgia alba samples were collected at a depth of 60 m in two climatic seasons, summer (September 2018) and winter (March 2019). For each sampling season, five apparently healthy individuals (biological replicate) of the sea octocoral L. alba were collected. Bacterial DNA extraction was performed using the Quick-DNA Fecal/Soil Microbe Kits Zymo Research. Amplification was carried out by PCR using the modified 16S Metagenomics Kit (Thermo Fisher Scientific) and DreamTaq Green PCR Master Mix (2X) (Thermo Fisher Scientific) protocols with two sets of primers: V2-4-8 and V3-6, 7-9. The amplicons were purified with, obtaining 10 samples for the preparation of the libraries. The libraries were constructed with the Ion Plus Fragment Library Kit protocol. The anneal was prepared with an emulsion PCR on the Ion One Touch 2 System (Life Technologies). The enriched anneal was loaded onto a PGM 318  chip, following the indications of the Ion PGM Hi Q View Sequencing Kit protocol for 400 bp fragments. Sequencing was performed in the Ion Torrent  Personal Genome Machine (PGM) of the National Laboratory for Plant Identification and Characterization (University of Guadalajara). The Ion Reporter  Software platform (ThermoFisher) detected and removed primers and short sequences (<150 base pairs) using the 16S Metegenomics Plugin (ThermoFisher). The sequences were grouped into operational taxonomic units (OTUs) with a 3% divergence, followed by the removal of OTUs with fewer than ten copies. The final OTUs were taxonomically classified using the curated databases of GreenGenes v13.5 and MicroSEQ (R) 16S Reference Library v2013.1 as references.