Agencourt ampure xp bead kit

Total DNA was extracted from EVs using DNeasy blood and Tissue Kits (QIAGEN Science, Hilden, Germany), following the manufacturer´s recommendations. The DNA was eluted into DNase/RNase-free water and its concentration and purity were evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Extracts were stored at −20 °C until used. Libraries and sequencing were performed as previously described [27 (link)]. Briefly, the amplification of V1-V3 regions of the 16S rRNA bacterial gene was performed using the primers 5′-GAGAGTTTGATYMTGGCTCAG-3′ forward and 5′-ACCGCGGCTGCTGGCAC-3′reverse with overhand adapters. Amplicons were purified using Agencourt AMPure XP bead kit (Beckman Coulter, Pasadena, CA, USA), indexed using Nextera XT index primers 1 and 2 (Illumina, San Diego, CA, USA), quantified by Quant-IT PicoGreen (Thermo Fisher Scientific, Waltham, MA, USA) and diluted to a concentration of 10 ng/μL. DNA samples were quantified by qPCR with KAPA 170 SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA). Samples were normalized, pooled and sequenced using Illumina MiSeq technology with v3 reagents (Illumina, San Diego, CA, USA), using paired end reads, with the GIGA Genomics platform (Liège, Belgium). Negative controls were used in the entirety for DNA extraction, library preparation and sequencing.

Total bacterial DNA was extracted from the stool samples with the PSP Spin Stool DNA Plus Kit 00310 (Invitek, Berlin, Germany), following the manufacturer’s recommendations.
PCR amplification of the 16S rDNA V1–V3 hypervariable region and library preparation were performed with the following primers (with Illumina overhand adapters), forward (50-GAGAGTTTGATYMTGGCTCAG-30) and reverse (50-ACCGCGGCTGCTGGCAC-30). Each PCR product was purified with the Agencourt AMPure XP bead kit (Beckman Coulter, Pasadena, CA, USA) and submitted to a second PCR round for indexing, using Nextera XT index primers 1 and 2. After purification, PCR products were quantified using the Quant-IT PicoGreen (ThermoFisher Scientific; Waltham, MA, USA) and diluted to 10 ng/µL. A final quantification of each library was performed using the KAPA SYBR^® FAST qPCR Kit (KapaBiosystems; Wilmington, MA, USA) before normalization, pooling and sequencing on a MiSeq sequencer using V3 reagents (Illumina; San Diego, CA, USA). Positive controls using DNA from 20 defined bacterial species and negative controls (from extraction and PCR steps) were included in the sequencing run [30 (link)].
Raw amplicon sequencing libraries were submitted to the NCBI database under bioproject number PRJNA682516.

Metagenomic DNA was extracted using the DNeasy® PowerSoil® kit (QIAGEN) with the following modification: 1 g (instead of 0.25 g) of the sample was used for the extraction. A control (no soil) was included during the processing to ensure that no contaminants were introduced during the isolation of metagenomic DNA. In order to determine actinobacterial diversity, the actinobacterial-specific 16S rRNA gene primer pair (Com2xf: 5′-AAACTCAAAGGAATTGACGG-3′; Ac1186r: 5′-CTTCCTCCGAGTTGACCC-3′) [18 (link)] was used, while fungal diversity was determined using the ITS1F (5′-CTTGGTCTTTAGAGGAAGTAA-3′) and ITS4Rv2 (5′-TCCTCCGCTTATTGATATGC-3′) primer pair [19 (link),20 ]. Illumina adapters and barcodes were incorporated by the high-throughput sequencing service provider, Inqaba Biotechnical Laboratories (Pretoria, South Africa). Amplicon libraries were purified using the Agencourt Ampure® XP bead kit (Beckman Coulter, Brea, CA, USA) and sequenced on an Illumina MiSeq™ instrument. Raw reads were pre-processed and supplied as .fastq files (NCBI BioProject accession number: PRJNA805212).