title: 16s rRNA of Human urine:Patient C02
design: A total of 19 metatranscriptomic samples were collected, of which 16 were selected for 16S ribosomal DNA (rDNA) amplification to enhance the specificity of microbial species identification. Urine samples were thawed, followed by the addition of 500 L of phosphate-buffered saline (PBS) to each tube. Samples were vortexed for 5 minutes to ensure homogeneity. DNA was then extracted from all samples using the DNeasy PowerSoil DNA isolation kit (Qiagen GmbH, Hilden, Germany), with a negative control included in each batch to monitor for potential contamination. DNA extracts were stored at 20C prior to downstream applications. The 16S rRNA gene was partially amplified using primers targeting the V3/V4 hypervariable regions, as per established protocols (reference). Amplified products were pooled in equimolar concentrations, purified using the MinElute Gel Extraction Kit (Qiagen GmbH, Hilden, Germany), and sequenced on an Illumina MiSeq platform (San Diego, CA, USA) using the MiSeq Reagent Kit V3 (600 cycles). To control for sequencing accuracy and contamination, the PhiX library served as a positive control, and a DNA isolation control was included as a negative control.