title: V3-V4 region of the 16S rRNA gene: Oyster-microbiome_preheatwave_subtidal_1
design: Digestive gland (~30 mg per sample) samples was homogenized (TissueLyser II, Qiagen) in 200 L of lysis buffer (ATL buffer with Proteinase K, Qiagen). Following that, DNA extractions were carried out using DNeasy Blood and Tissue kits. 16S rRNA gene amplicon sequencing (targeting 16S V3-V4 regions; 341F 5-CCTACGGGNGGCWGCAG-3, 785R 5-GACTACHVGGGTATCTAATCC-3) of DNA from digestive gland was carried out generally following the 16S Metagenomic Sequencing Library Preparation Manual (#15044223). Amplicon PCR was carried out with an iProof High-Fidelity PCR Kit (#1725330, Bio-Rad Laboratories) in 20-L reaction volumes comprising 10 L of iProof HF Master Mix, 2 L of gDNA, 6 L of molecular grade water, and 1 L of 10 M 16S primers (with overhang adapters). Index PCR was carried out using an iProof High-Fidelity PCR Kit (#1725330, Bio-Rad Laboratories) in 40-L reaction volumes comprising 20 L of iProof HF Master Mix, 5 L of gDNA, 5 L of molecular grade water, and 5 L of Nextera XT Index Primers. All PCR cleanup was carried out using QIAquick PCR Purification Kits (#28104, Qiagen), quantified using a NEBNext Library Quant Kit. Indexed samples were diluted to 2 nM and pooled prior to sequencing.