Kapa 170 sybr fast qpcr 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.

Libraries and sequencing were performed as previously described [42 (link)]. Briefly, amplification of the 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 a 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 by GIGA Genomics platform (Liège, Belgium). A bacterial community composed of known proportions of Carnobacterium maltaromaticum, Lactococcus lactis subsp. cremoris, Leuconostoc carnosum, Pseudomonas aeruginosa and Streptococccus thermophilus was used as a positive control. Negative controls were used in their entirety for DNA extraction, library preparation and sequencing.