Sequalprep

Plants were harvested after 22 days of growth; shoots and roots were collected (roots were washed), dried at 50 °C for 48 h and weighed. Shoot biomass was measured for each individual separately whereas root biomass was characterised at the pot scale.
Soil mineral nitrogen content (nitrate and ammonium) at 22 days was measured by shaking 10 g of soil in 50 ml of KCl for 1 h, decantation for 45 min and analysis of the filtered supernatant with the analyser Global 240 (BPC Biosed, Rome, Italy).
Considering the colonization of the pots by plant root systems, we could hypothesize that the soil was under the influence of the plants in the entire pot. Thus, the soil of each pot was mixed and DNA was extracted from a sample of 280 mg (DNeasy PowerSoil Kit, Qiagen, Venlo, Netherlands).
After ensuring that there was no PCR inhibition, 16S rRNA gene was amplified using the Pro341F/Pro805R couple of primers (targeting Bacteria, Takahashi et al. 2014) (link). A second PCR allowed the barcoding of the samples by hybridization of specific couples of primers on the adapters carried by the Pro341F/Pro805R primers. PCR products were normalized (SequalPrep, ThermoFisher Scientific, Waltham, USA), pooled and sequenced on Illumina MiSeq (Microsynth, Balgach, Switzerland).

The indexing PCR reactions were then purified and normalized using a SequalPrep normalization plate (Thermo Fisher Scientific), followed by elution in 20 µl of elution buffer. An even volume of the normalized libraries was pooled and concentrated using 1x AmpureXP beads (Beckman Coulter).
Pooled libraries were quantified using a Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific), fragment sizes were assessed using an Agilent Bioanalyzer High Sensitivity assay, and libraries were normalized to 2 nM for sequencing. The library was denatured with NaOH and prepared for sequencing according to the protocols described in the Illumina MiSeq Denature and Dilute Libraries Guide and sequenced in a portion of a MiSeq 600 cycle v3 run.

Library preparation and sequencing of the amplicons were carried out at the Core Facility Molecular Biology, Center for Medical Research at the Medical University Graz, Austria. In brief, DNA concentrations were normalised using a SequalPrep™ normalisation plate (Invitrogen), and each sample was indexed with a unique barcode sequence (8 cycles index PCR). After pooling of the indexed samples, a gel cut was carried out to purify the products of the index PCR. Sequencing was performed using the Illumina MiSeq device and MS-102-3003 MiSeq® Reagent Kit v3-600cycles (2x150 cycles). The obtained fastq data is available in the European Nucleotide Archive under the study accession number: PRJEB41618.
The fastq data analysis was performed using QIIME2 [43] as described previously [44] . After quality filtering, the DADA2 algorithm [45] was used to denoise truncated reads and generate amplicon sequence variants (ASVs). Taxonomic classification [46] was based on the SILVA v132 database [47] and the obtained feature table and taxonomy file were used for further analysis (Supplementary Tab. 4). The overlapping features from negative controls (DNA extraction and PCR negative controls) were manually subtracted or removed from both the bacterial and archaeal dataset. The reads classified as chloroplast and mitochondria were also removed.