GSM9127816: PL_074_EV; Homo sapiens; miRNA-Seq
Samples obtained by SEC for RNA extraction were concentrated using Vivaspin Turbo 4 centrifugal concentrators with 10 kDa molecular weight cut-off (Sartorius). A fresh device was used for each sample, loaded with an equal volume and spun at 4,000 g for 1 hour at 4°C. Samples were transferred immediately to a 1.5 mL Eppendorf safe lock microtube and weighed using a fine weighing scale. These values were plotted against a standard curve generated using PBS and an estimated volume calculated for subsequent reactions. Total RNA from plasma samples (total and EV) were extracted using the phenol-free miRNeasy Serum/Plasma advanced kit according to manufacturer's instructions. Stations were prepared for RNA extraction using RNaseZap RNase decontamination solution (Thermo Fisher Scientific). Lysis involved addition of proprietary Buffer RPL at a volumetric ratio of 10:3 and vortexed before incubation at RT for 3 minutes. 1 µL of Buffer RPP per 3 µL of Buffer RPL was added, vortexed for 20 seconds and incubated at RT for 3 minutes. Samples were centrifuged at 12,000 x g for 3 minutes at RT to obtain a pellet and an equal volume (Sample Volume + Buffer RPL + Buffer RPP) of isopropanol added to the supernatant. Up to 700 µL of the sample was transferred to an RNeasy UCP MinElute spin column (QIAGEN) and centrifuged at 8,000 x g for 30 seconds at RT. This was repeated using the remainder of the sample in 700 µL aliquots. Optional on-column DNase digestion was not performed. 700 µL of Buffer RWT was added to the RNeasy UCP MinElute spin column and centrifuged at 8,000 x g for 30 seconds at RT with the flow-through discarded. 500 µL of Buffer RPE was then added to the RNeasy UCP MinElute spin column and centrifuged at 8,000 x g for 30 seconds at RT with the flow-through discarded. Finally, 500 µL of 80% ethanol was added to the RNeasy UCP MinElute spin column and centrifuged at 8,000 x g for 2 minutes at RT with the flow-through discarded. RNA remaining in the spin column was transferred to a fresh 2 mL collection tube after drying the membrane by spinning the column with the lid open at 16 000 x g for 5 minutes. RNA was eluted with 14 µL of RNase-free water and used immediately, or frozen at -80°C until required. RNA samples were prepared for small RNA sequencing using Qiagen's QIAseq small RNA Library Prep kit according to manufacturer protocol. 5 µL of the RNA eluate was used in low input samples (i.e. EV-RNA). The finished libraries were quality controlled using an Agilent Bioanalyzer 2100 and quantified by use of qPCR. Libraries were pooled and sequenced on an NextSeq 500 system (Illumina, San Diego, USA) using the default single-end 75 base pair protocol to include integrated unique molecular indices (UMIs).