title: GSM6705905: 20118a041; Homo sapiens; RNA-Seq
library_construction_protocol: RNAs were extracted from freshly purified EVs without any freeze/thaw steps in between. To this end, 350 μl of lysis buffer from the SeraMir Exosome RNA Amplification kit (System Biosciences) was added to the purified EVs, vortexed for 15 sec at maximum speed, incubated for 5 mins at room temperature and 200 μl of 100% ethanol (RNA grade) added. The sample was vortexed for 10 sec at maximum speed and the precipitating solution was transferred to an ExoRNA spin column. Samples were bound and washed twice (each 400 µl of wash buffer) with centrifugation steps of 1 min at > 15.000 g. Flow-throughs were discarded. Columns were dried 2 min at > 15.000 g and placed into a new DNA low-bind tube (Eppendorf). 23 μl of RNase free water was directly applied onto the middle of the column without touching it and incubated for 1 min at room temperature. RNAs were eluted by centrifugation for 2 min at < 400 g, followed by centrifugation for 1 min at > 15.000 g. RNA was aliquoted, frozen in liquid nitrogen and stored at -80°C. RNA quality assessment and quantification were carried out using a Agilent 2100 Bioanalyzer with RNA 6000 pico chips (Agilent Technologies). The sequencing library was prepared using the SMARTer smRNA-Seq Kit (Takara Bio, Kusatsu, Japan) with 100 pg total RNA as input according to the manufacturer's protocol. In short, adenylation of the 3' ends enabled priming of the reverse transcription reaction using an oligo(dT) primer containing the Read 2 adapter sequence for sequencing. Subsequently, cDNA synthesis was performed, making use of the template-switching technology and adding the Read 1 sequencing adapter. Libraries were then amplified in a PCR with 21 cycles using barcoded primers. Eight differently barcoded forward (i5 index) primers and twelve different reverse (i7 index) primers provided in the kit were used in 66 different combinations to enable multiplexing of all 66 samples on one flowcell. After the PCR, samples were cleaned up using the column-based NucleoSpin Gel and PCR Clean-Up kit (Macherey-Nagel, Düren, Germany). The double two-sided size selection described in the user manual was omitted to also retain larger molecules. The molarity of the libraries was assessed using the High Sensitivity NGS assay of the FragmentAnalyzer (Agilent, Santa Clara, USA) for average fragment size, and fluorometric quantitation to measure library concentration with the dsDNA High Sensitivity assay on a Qubit 3.0 (both Thermo Fisher Scientific, Waltham, MA, USA). The libraries were pooled in an equimolar fashion, denatured according to the manufacturer's instructions and diluted to 270 pM. Sequencing was performed on a NovaSeq 6000 (Illumina, San Diego, CA, USA) as 150 bp paired-end reads with an average sequencing depth of 11 Mio clusters (i.e. read-pairs) per sample.