Metagenomic- coffee fermentation at 24 hr middle layer-Euk
DNA was extracted from samples collected at 0, 12, 24, 36, 48, and 72 h from the bottom, middle and top layers using Power Soil Kit (Qiagen). DNA was quantified with a Nanodrop spectrophotometer (Thermo Scientific). The variable regions V3-V4 of the 16S rRNA gene of bacteria and the ITS region of fungi were amplified from the extracted total DNA using primers 341F/805R and 3F/805R, respectively, and both barcoded with Nextera indexes, according to manufacturer instructions (Illumina). The amplicons were quantified with Qubit DNA HS kit (Thermo) and sequenced with MiSeq Reagent 500v2 kit (Illumina), in paired 2x250b. The fastq files were filtered and demultiplexed with bcl2fastq (Illumina). Taxonomic identification was performed with QIIME2.