GSM4065058: 5pre; Homo sapiens; miRNA-Seq
Total RNA from purified EVs was isolated using miRCURY RNA isolation kit - plasma and serum (Exiqon) and eluted in 100 µl RNase free water. To concentrate RNA, 10 µl 3M, pH 5.5 Sodium Acetate, 250 µl pre-chilled 99% ethanol and 1 µl Glycoblue (Ambion, MA, USA) were added and incubated at -20°C overnight. RNA was pelleted by centrifugation at 16,000g for 10 min at 4°C and re-suspended in 6 µl RNase free water The small RNA library of EVs was constructed using Illumina TruSeq Small RNA Sample Prep Kit (Illumina, CA, USA) by adding 5 µl of total RNA as input. Due to lower RNA input compared to the standard protocol, the amount of adaptors was reduced to 1/10, the other reagents were reduced by half, and PCR cycles were increased from 12 to 15. The library was purified in the size range 140bp-160bp by Pippin Prep (Sage Science, MA, USA). The size and purity of the cDNA libraries were validated on a 2100 Bioanalyzer High Sensitivity DNA chip (Agilent, CA, USA) and the concentration was quantified using KAPA Library Quantification Kit (KAPA biosystems, Switzerland) The libraries were pooled as required and sequenced on the Illumina Nextseq 500 instrument by Exiqon
GEO Accession
GSM4065058