Concentrator plus 5305 vacuum centrifuge

EV sample was concentrated using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany), and protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Scientific). Concentrated sEV preparations and lysate of HCT116 cells were lysed in Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific), heated for 5 min at 95 °C, and subjected to electrophoresis using 10% SDS-PAGE. Proteins were transferred to an Immobilon-P PVDF Membrane (Merck Millipore) and the excess protein binding sites on the membrane were saturated with 5% bovine serum albumin blocking buffer (1 × TBS, 0.1% Tween-20) for 1 h. The membrane was incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-CD81 (1:250, mouse, catalogue number sc166029), anti-CD63 (1:300, mouse, sc5275) from Santa Cruz Biotechnology, anti-Alix (1:20000, rabbit, ab186429) from Abcam, anti-TSG101 (1:200, mouse, 612696) from BD Biosciences, and anti-Calnexin (1:1000, rabbit, 2679) from Cell Signaling. After incubation, the membrane was washed three times with 5% TBS-Tween and then, incubated with peroxidase-labelled secondary antibody (Santa Cruz Biotechnology) for one hour. After three washes, immobilized proteins were detected utilizing Clarity Western ECL Substrate (Bio-Rad) and the UVITEC chemiluminescence imager (UVITEC Cambridge, UK).

Isolated and undiluted RNA was concentrated from 50 to 5 μl using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany). RNA was not further fragmented or subjected to any kind of selection. Sequencing libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, USA) according to manufacturer’s recommendations with two exceptions; due to low RNA input, libraries were subjected to 18 cycles of amplification, and NEBNext Adaptor was diluted 200× before ligation. Libraries were individually barcoded with NEBNext Multiplex Oligos for Illumina (New England Biolabs, USA). Library concentration and quality were assessed fluorometrically using Qubit 4.0 Fluorometer and Qubit HS DNA Assay Kit (Thermo Fisher Scientific) and electrophoretically using Agilent 2200 TapeStation System and High Sensitivity D1000 ScreenTape (Agilent Technologies). Each library was diluted to a final concentration of 4 nM and pooled equimolar prior to clustering. RNA sequencing (single read, 75 cycles) was performed using the NextSeq 500/550 High Output Kit v2 and the NextSeq 500/550 instrument (Illumina, USA).