GSM8710136: miRNA profile subject 178; Homo sapiens; miRNA-Seq
Total RNA from saliva samples was extracted using the Maxwell® RSC miRNA Tissue (Promega, Milan, Italy) following the manufacturer's instructions. Initially, 400 µL of saliva samples were added to chilled 200 μl of 1-Thioglycerol per ml of Homogenization Solution and following the instructions of manufacturer. At the end of the automated extraction, RNA samples were eluted in 60 µL of water. RNA concentration was measured with a Qubit fluorometer using Qubit microRNA assay (Thermofisher, Milan, Italy). NEBNext Multiplex Small RNA Library Prep for Illumina kit (New England Biolabs, Inc) was used to convert small RNA transcripts into barcoded complementary DNA (cDNA) libraries. For each library, 100 ng of RNA was processed as starting material. Each library was prepared with a unique indexed primer. Multiplex adapter ligations, RT primer hybridization, RT reaction, and PCR amplification were performed according to the manufacturer's protocol. After PCR amplification, the cDNA constructs were purified with the Monarch PCR & DNA cleanup kit (New England Biolabs, Inc), following the modifications suggested by the NEBNext Multiplex Small RNA Library Prep for Illumina protocol. Final libraries were loaded on the Bioanalyzer 2100 (Agilent Technologies) using the DNA High Sensitivity Kit (Agilent Technologies) according to the manufacturer's protocol. Libraries were pooled together (in 35-plex or 40-plex) and further purified with a gel size selection. A final Bioanalyzer 2100 run with the High Sensitivity DNA Kit (Agilent Technologies) allowed to assess DNA library quality regarding size, purity, and concentration.