GSM8625571: Healhy Subject; Homo sapiens; miRNA-Seq
Culture medium was centrifuged twice (Sigma 2-16PK, Merck, NJ, USA) at 2000 rpm for 10 minutes at room temperature to remove the bulk of the cell debris. The supernatant was then ultracentrifuged (LE-80K, Beckman Coulter, CA, USA) at 17300 rpm for 20 minutes at 4°C (Ti50 E4280 rotor, Beckman Coulter, CA, USA) and 4000 rpm for 120 minutes at 4°C (Ti80 1377 rotor, Beckman Coulter, CA, USA). After discarding the supernatant, the pellets were resuspended in 1 ml 0.22-µm-filtered PBS. Finally, the samples were subjected to ultracentrifugation at 4000 rpm for 120 minutes at 4°C (Ti80 1377 rotor, Beckman Coulter, CA, USA) to obtain pellet particles. Half of the particles were resuspended in 200 µL PBS for exosome characterization by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and flow cytometry. The rest were resuspended in 800 µL QIAzol lysis reagent (Qiagen, Hilden, Germany) for RNA extraction. Total RNA was extracted using the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Two microliters of the remaining RNAse-free water were kept for quantification by Quant-IT RiboGreen RNA assays kit (Thermo Fisher Scientific, MA, USA). The RNA was stored at -80°C prior to sequencing. Maximum storage time of RNA samples was 3 months. The library preparation was done using the QIAseq miRNA Library Kit (QIAGEN). A total of 10ng total RNA was converted into miRNA NGS libraries. After adapter ligation, UMIs were introduced in the reverse transcription step. The cDNA was amplified using PCR (19 cycles) and during the PCR indices were added. After PCR the samples were purified. Library preparation was quality controlled using capillary electrophoresis (Tape D1000). Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pool(s) were quantified using qPCR. The library pool(s) were then sequenced on a NextSeq (Illumina Inc.) sequencing instrument according to the manufacturer instructions (1x75, 2x10). Raw data was de-multiplexed and FASTQ files for each sample were generated using the bcl2fastq2 software (Illumina inc.).