GSM5082149: 8 days after surgery control (sham) sample 3 [8dsham3]; Mus musculus; RNA-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. To detect coding and long non-coding RNAs present in EVs, the TruSeq stranded mRNA library preparation kit (Illumina) was used, following the manufacturer's instructions but skipping the depletion of ribosomal RNAs and purification of the polyA+ fraction phases. Briefly, 40 ng of total RNA extracted from EVs were fragmentated for 8 min at 94°C. First strand and second strand cDNA were synthetized, a single A nucleotide was added to the 3' ends of the blunt fragments, and multiple indexing adapters were ligated at the end of the double strand cDNA. A 15 cycles PCR was performed to selectively enrich those DNA fragments with adapter molecules on both ends. Each library was analysed with the DNA 1000 chip (Agilent) using Agilent 2100 Bioanalyzer and quantified using the Qubit DNA HS kit (Life Technologies). A pool of 12 libraries (pooled at equimolar concentration) was generated, quantified with Qubit DNA HS kit and run at the concentration of 1.6 pM on the NextSeq500 (Illumina) sequencer in 75 nts paired end sequencing mode following the manufacturer's instructions.
GEO Accession
GSM5082149