title: miRNAseq of osteosarcoma:Biopsy_45
design: RNA was isolated with the QIAGEN RNeasy Plus Universal Mini Kit (Qiagen) using an adapted protocol designed to increase the quality of extracted RNA from bone. RNA quantity was determined on the Qubit using the Qubit RNA High Sensitivity Assay Kit (Life Tech) and RNA quality was determined on the Bioanalyzer using the RNA Pico Kit (Agilent). Using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (NEB), 100ng of total RNA was converted into a DNA library following the manufacturers protocol, with no modifications. Library quantity was determined using the Qubit High Sensitivity DNA Kit (Life Tech) and library size was determined using the Bioanalyzer High Sensitivity Chip Kit (Agilent). Finally, libraries were subjected to qPCR using the Universal Library Quantification Kit for Illumina (Kapa Biosystems) and run on the 7900HT Fast qPCR machine (ABI).  Libraries passing quality control were diluted to 2nM, combined into library pools, and sequenced on the NextSeq 500 (Illumina) at a final concentration of 2pM. Sequencing was done across three NextSeq Single Read 75 Cycle High Throughput V2 flowcells following standard protocols.