GSM5082164: 8 days after surgery control (sham) sample 6 [8dSham626]; Mus musculus; miRNA-Seq
Blood was collected into K2 EDTA-coated S-Monovette® by cardiac puncture. Plasma was prepared within 1 hour of blood sampling by centrifuging at 1000g for 10 mins at 4°C followed by a second centrifugation of the supernatant at 2000 rpm for 15 mins and frozen at -80°C till further processing. Circulating EVs were collected from 250 µl of plasma. Briefly, plasma was thawed on ice and 63 µl of ExoquickTM (System Biosciences) was added to EV preparation according to the maunfacturer's instructions. For total RNA purification, EVs pellets were resuspended in 200 µl PBS and 1 ml of QIAzol Lysis reagent (Qiagen) added. RNA was extracted using miRNeasy serum/plasma kit (Qiagen) in an automated way using the QIAcube instrument (Qiagen), thus preventing operator-depending variability. Total RNA was estimated quantitatively with Qubit RNA HS kit (ThermoFisher Scientific) and then stored at -80°C till further use. Small RNA libraries were prepared using Small RNA Library Preparation Kit NebNext (New England Biolabs) according to manufacturer instructions starting from 40 ng of EV-derived RNA and using 15 PCR cycles. Each library was analysed with the High Sensitivity DNA chip (Agilent) using Agilent 2100 Bioanalyzer. Pools of 12 libraries were concentrated with AMPure XP magnetic beads (Beckman Coulter) and eluted in 40 µl of nuclease free water. The eluate from each pool was loaded onto a 6% precast gel (Life Technologies) and a ≈146 bp band was cut and put in 300 µl of nuclease free water overnight in 2ml low bind eppendorf tube. Gel debris were removed with Qiaquick gel extraction kit (Qiagen) and cDNA was precipitated using 3M NaOAc, 100% cold ethanol and glycogen, centrifuged at 20 000g for 20 mins at 4°C. After one wash with 70% ethanol, cDNA was resuspended in 10 µl nuclease free water and quantified using the Qubit DNA HS (Life Technologies). Pool was analysed with the High Sensitivity DNA chip (Agilent) using Agilent 2100 Bioanalyzer. Two pools (2 x 12 samples purified indexed libraries) were run at the concentration of 1.6 pM on the NextSeq500 sequencer (Illumina) according to the manufacturer's instructions, in 75 nts single end sequencing mode.
GEO Accession
GSM5082164