GSM8595683: Car-1 rep2 RNA; Homo sapiens; RNA-Seq
Ultrafiltration was performed prior to SEC. SEC with the Automated Fraction Collector (AFC-V1, Izon) was performed to isolate vesicles using 2 qEV original / 70 nm gen 2 columns (IZON) per sample. Using a buffer volume of 2.70 ml, and a fraction volume of 0.5 ml, the particles of interest were collected in fractions 3 and 4 (between 1 and 2 ml of the Purified Collection Volume (PCV)). Both fractions of the 2 isolations were pooled, resulting in a volume of 2 ml per sample. 1 ml of the vesicle isolate was concentrated with an Amicon Ultra-2 Centrifugal Filter Unit (Millipore) to 250 µl. Total RNA from the EVs was isolated using the miRNeasy serum/plasma kit (QIAgen) according to the manufacturers' protocol with minor adjustments. mRNA libraries were prepared using the SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara Bio Inc.) according to the manufacturers' protocol, including ribosomal cDNA depletion