16S rRNA of negative extraction 1
A no sample (null) DNA extraction to control for possible kit/lab contaminants. For this, 400 L of AP1 buffer + RNase A were used for DNA extraction with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions.The yield and quality of all extractions was assessed using a Nanodrop 2000 (ThermoFischer, Waltham, MA, USA). For the characterization of the overall prokaryotic community composition, the hypervariable regions V5 and V6 of the 16S rRNA gene were amplified using the primers  784F  5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGATTAGATACCCTGGTA-3 and 1061R 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCRRCACGAGCTGACGAC-3 (Illumina overhang adaptor sequences are underlined) [43, 44]. PCRs were run in triplicate using a reaction volume of 10 L with 5 L of Qiagen multiplex PCR master mix, 10 ng of DNA template and final primer concentrations of 0.5 M, respectively. PCR cycling conditions consisted of an initial activation step at 95 C for 15 min, followed by 27 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 30 s, followed by a final extension step at 72 C of 10 min. Successful amplification of all samples was confirmed by gel electrophoresis before products of triplicate PCRs for each sample were pooled. PCR products were subjected to an indexing PCR (8 cycles) using the Nextera XT Index Kit v2 (Illumina) according to the manufacturers instructions. Indexed samples were then cleaned and normalized using the SequalPrep Normalization Plate Kit (Invitrogen, Carlsbad, CA, USA), pooled at equimolar ratios and verified on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA, USA). Amplicon libraries were sequenced at the KAUST Bioscience core laboratory on the Illumina MiSeq platform using 2  300 bp overlapping paired-end reads with 20% spiked-in phiX.