Colorectal cancer–derived extracellular vesicles selectively enrich and transfer functional miR-1290 into parental cells
Description
MicroRNA sequencing data of miRNA samples isolated from colorectal cancer (CRC) cell lines: HCT116, SW480 and SW620, and normal CCD841CoN cell line, and extracellular vesicles derived from these cell lines.
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Extracellular vesicles were isolated from cell culture supernatants. Supernatants were centrifuged at 500 × g for 10 min and 3,200 × g for 15 min to remove debris, then ultracentrifuged at 100,000 × g for 2 h using a Sorvall™ WX+ Ultracentrifuge with a T-1270 rotor (Thermo Fisher Scientific, Waltham, MA, USA). TEV pellets were washed twice with filtered PBS and stored at –80C for further analysis. TEVs were regularly tested for endotoxin contamination by the Limulus test (Pierce Chromogenic Endotoxin Quant kit, Thermo Fisher Scientific) according to the manufacturer’s instruction. Total RNA was isolated from cells and EVs using the mirVana™ miRNA Isolation Kit and the Total Exosome RNA & Protein Isolation Kit, respectively (Invitrogen, Life Technologies and Macherey-Nagel, Dueren, Germany) according to manufactures protocols. TEV-derived RNA was concentrated to 10 μL using the GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Fisher Scientific). miRNA expression profiling was performed by TAmiRNA GmbH (Vienna, Austria) using small RNA libraries prepared with the RealSeq Biofluids kit (RealSeq Biosciences, Santa Crus, CA, USA). RNA input included 85 ng for cellular RNA and 8.5 μL of EV RNA; 1 μL of spike-in control was added to each sample. Libraries were amplified with 21 or 24 PCR cycles (cells and EVs, respectively), quality-checked using the Agilent DNA1000 chip, and sequenced on the Illumina NextSeq 550 platform (75 bp single-end reads). Data were processed using the miND pipeline: quality control (FastQC v0.11.8, MultiQC v1.7), adapter trimming (Cutadapt v2.3), alignment (Bowtie v1.2.2, miRDeep2 v2.0.1.2), and annotation using GRCh38.p12 and miRBase v22.1.
Institutions
- Uniwersytet Jagiellonski w Krakowie Collegium Medicum