GSM9349433: Q_12114_S7_R1_001; Homo sapiens; RNA-Seq
Unstimulated whole saliva was collected in the morning (9:00–10:00 AM) from healthy male volunteers (18–40 years old) who had refrained from eating, drinking, or smoking for at least 1 hour. Participants passively drooled into sterile tubes, and a minimum of 4 mL was collected per individual. Samples showing visible blood contamination were excluded. Saliva was placed on ice and processed within 1 hour: first centrifuged at 300 × g for 10 min at 4 °C to remove cells, followed by 3000 × g for 30 min at 4 °C to eliminate residual debris. The resulting supernatant was used for EV isolation. Total small RNA was extracted from EV and WS samples using the miRNeasy Serum/Plasma Kit (Qiagen, #217184) and eluted in 14 µL of RNase-free water. RNA yield was quantified using the Qubit™ microRNA Assay Kit (ThermoFisher, #Q32881), and size distribution was assessed via the LabChip GX system (Caliper-PerkinElmer) using the Small RNA Assay (#CLS153530) to confirm the presence of miRNAs. Small RNA libraries were prepared from 10 ng of miRNA using the TruSeq Small RNA Library Prep Kit (Illumina, #RS-200-0012). After adapter ligation, cDNA synthesis, and PCR amplification (11 cycles), libraries were purified with Ampure XP beads (Beckman Coulter, #A63880), assessed on the LabChip GX system, and quantified via Qubit™ dsDNA HS Assay (#Q32851). Equimolar pooling was followed by sequencing on the NextSeq 500 platform using the high output v2.5 kit (1.8 pM, #20024906).