title: RNA-Seq of colorectal: BC
design: Peripheral blood samples from individuals were collected in EDTA tubes following a regular venipuncture procedure. After centrifugation at 3,000 g for 15 min at 4 C, the plasma was aspirated and stored at -80 C before use.A total of 3 mL plasma was collected from each patient, and each plasma sample was divided into two aliquots (2 mL for sEV isolation, 1 mL for direct RNA isolation). The ultracentrifugation (UC) method was optimized according to the method previously described. After thawing at 37 C, plasma samples were centrifugated at 3,000 g for 15 min to remove cell debris. Then, the supernatant was diluted by seven-fold volume of PBS, centrifuged at 13,000 g for 30 min, and processed through a 0.22 m filter to remove large particles. The supernatant was ultracentrifuged using a P50A72-986 rotor (CP100NX; Hitachi, Brea, CA, USA) at 150,000 g, 4 C for 4 h to pellet the exosomes. The pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged again at 150,000 g 4 C for 2h. After PBS washing, the exosomes pellet was re-suspended in 100 l PBS. Total RNA was extracted and purified from plasma exosome using miRNeasy Mini kit (Qiagen, cat. No. 217004) according to standard protocol. RNA degradation and contamination, especially DNA contamination, was monitored on 1.5% agarose gels. RNA concentration, purity, and integrity were assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). For small RNA libraries, a total amount of 2.5 g RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following manufacturers recommendations and index codes were added to attribute sequences to each sample. At last, PCR products were purified (AMPure XP system) and library quality was assessed with both the Agilent Bioanalyzer 2100 and qPCR.