Two supernatant removal steps were used on 3 ml of culture media to get rid of the un-precipitated material. EV lysis and RNA isolation was performed on 500 µl of media using exoRNeasy Serum/Plasma Maxi Kit (QIAGEN). Library preparation was done using the QIAseq miRNA Library Kit (QIAGEN). Out of 12µl of isolated RNA, 5µl was converted into microRNA NGS libraries. Adapters containing 12nt-long unique molecular indices (UMIs) were ligated to the RNA. Then RNA was converted to cDNA. The cDNA was amplified using PCR (22 cycles) and during the PCR indices were added. After PCR the samples were purified. Library preparation quality control was performed using either Bioanalyzer 2100 (Agilent) or TapeStation 4200 (Agilent). Based on the quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pools were quantified using the qPCR ExiSEQ LNA™ Quant kit (Exiqon). The library pools were then sequenced on a NextSeq500 sequencing instrument according to the manufacturer instructions using 1x75bp reads.
GEO Accession
GSM3405414