GSM4907702: O5; Homo sapiens; RNA-Seq
Samples were thawed at room temperature and filtered with 0.8 μm Millex AA syringe filters (Merck Millipore) to exclude larger particles, such as remaining platelets, while retaining exosomes and MVs. Total RNAs were extracted from 0.3 ml of plasma with miRNasy micro kit (Qiagen) using a modification, where Trizol LS (Life Technologies) was used instead of the Qiazol reagent according to manufacturer's instructions. RNA consisted of total RNA, miRNA-enriched fraction was not purified separately. RNAs were eluted with 14 μl of RNAse-free water and stored in Low DNA binding Eppendorf tubes (Eppendorf) at −80 °C. The integrity of total RNA in the samples was evaluated using Agilent Bioanalyzer RNA Nano/Pico chips (Agilent). Qubit fluorometer was used for the RNA quantitation, along with Qubit RNA-kit (Life Technologies). ScriptSeq Complete Gold System (Epicentre) was used to ripodeplete rRNA from the 1 μg of total RNA, and this ripodepleted RNA was then used for RNA-seq library preparation. The produced RNA-seq libraries were purified using SPRI beads (Agencourt AMPure XP, Beckman Coulter), and their quality control was evaluated on High Sensitivity chips using an Agilent Bioanalyzer (Agilent).