GSM3937068: EVs_A2; Mus musculus; miRNA-Seq
EVs were isolated by differential centrifugation (130 x g for 10 min, 10 000 x g for 30 min, 100 000 x g for 2 h). Pellet or cells were mixed with QIAzol and small RNA was isolated using the miRNeasy micro Kit (Qiagen). RNA was size-selected between 15 to 200 nt on 10% TBE-urea gel. Gel-purified RNA was eluted overnight in 0,3 M NaCl and then precipitated with 100% isopropanol and Glycoblue for 1 h at -20°C. The pellet was washed once with 75% ethanol and dissolved in nuclease-free water. The purified fraction was confirmed by Bioanalyzer Small RNA chip (Agilent). Library preparation was based on the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs) with slight modification. In brief, RNA was first ligated to the 3' adapter and then to the 5' adapter, both of which contained 4 random bases and were chemically synthesized by Bio Scientific. Adapter-ligated RNA was reverse-transcribed and PCR-amplified for 14 cycles using index primers. The PCR-amplified cDNA construct was purified using AMPure XP beads (Beckman Coulter) and was checked on the Bioanalyzer using High Sensitivity DNA chip (Agilent). Size selection of the RNA library was performed on LabChip XT instrument (Perkin Elmer) using DNA 300 assay kit in the range 140-300 bp, the libraries were pooled in equal molar ratio and sequenced on Illumina HiSeq 2500 for 51 cycles plus 7 cycles index read.
GEO Accession
GSM3937068