GSM3263379: CMT-27; Canis lupus familiaris; miRNA-Seq
RNA was extracted from 5 mL of cell-free, serum-free conditioned media using the Norgen Biotek Urine/Cell Culture Exosomal RNA Isolation kit (Norgen Biotek, Thorold, ON, Canada) according to manufacturer instructions. After the lysis step, 10 pM final concentration synthetic miRVana cel-miR-39-3p mimic (Thermo Fisher Scientific, Waltham, MA, USA) was spiked into samples as an external control for technical variation. Extracted RNA was stored at -80°C until being shipped on dry ice to the Genomic Services Laboratory at the Hudson Alpha Institute for Biotechnology. Briefly, total RNA (500 ng) from each sample was used for miRNA library preparation. For miRNAseq, the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs Inc., Ipswich, MA, USA) was followed according to the manufacturer's protocol. Final  miRNA library quantities were assessed using a Qubit® 2.0 Fluorometer and library quality was determined using a DNA High Sense chip on Agilent 2100 Bioanalyzer. Further library quantification was completed using the qPCR-based KAPA Biosystem Library Quantification kit (Kapa Biosystems Inc., Woburn, MA, USA). For miRNAseq, individual sample libraries were diluted to a final concentration of 1.25 nM. Equimolar amounts of individual barcoded  samples from each group were pooled prior to sequencing. miRNAseq was conducted using an Illumina HiSeq 2500 instrument (Illumina Inc., San Diego, CA, USA) at 50bp Single End condition generating approximately 15 million reads per sample.