RNA-seq of bacteria in Toxopneustes roseus: adult sea urchin
The samples were obtained in the summer and winter seasons of two consecutive years (2017-2018 and 2018-2019). For each sampling site and season, three apparently healthy individuals (replicates) of the sea urchin T. roseus were collected (72 replicates). Each replicate sample included pedicellars, spines, digestive tract and gonads. Bacterial DNA extraction was performed using the modified TRIzol Reagent protocol (Thermo Fisher Scientific). Prior to amplification, the three replicates from each sampling site, season, and year were adjusted to the same concentration and pooled (24 samples). 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 (48 amplicons) were combined and purified with, obtaining 24 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.