EV miRNA-Seq of homo sapiens: adult female plasma isolated by ExoQuick
Subjects and sample collection. Samples of the study subjects were obtained in healthy subjects and T2DM patients with DKD. Study participants were instructed to collect their entire early morning urine and plasma on the day of the study and to present the samples to the laboratory. The urine and plasma samples from each patient were aliquoted at 50 mL and 1 mL per tube, respectively. The tubes were stored at -80C until further use. All protocols and procedures were approved by the Gachon University Gil Medical Center Institutional Review Board (#2016-195) and written informed consent was obtained from all participants. This study adheres to the ethical rules of the declaration of Helsinki and was registered at Clinical Research Information Service (CRIS, https://cris.nih.go.kr) (registration number KCT0003573) in accordance with the WHO International Clinical Trials Registry Platform.   Preparation for EV isolation  Urine samples from participants were thawed overnight at 4C. After vortexing for 90 seconds, the samples were centrifuged at 2,000 g for 15 min to pellet cellular debris. A total of 400 mL of urine sample from each participant was used to isolated EVs by using the ultracentrifugation (UC) (100 mL of urine). A total of 1 mL of the plasma sample from each participant was used to isolate EVs by UC. The isolated EVs were used in the tests which included EV counting, immunoblotting analysis, and microRNA profiling by NGS. Ultracentrifugation (UC) The cell-free supernatant was centrifuged at 13,500 g for 20 min at 4C to remove large vesicles. Then, the supernatant was filtered through a 0.22 m pore size filter, transferred to new UC tubes and centrifuged at 200,000 g for 65 min at 4C to pellet EVs using a Optima L-100 XP ultracentrifuge with type 70 Ti fixed-angle titanium rotor (Beckman Coulter, Indianapolis, IN, USA). The exosome pellet was resuspended in 400 l of phosphate-buffered saline (PBS). EV precipitation using ExoQuick-TC reagent. EV precipitation reagent ExoQuick-TC Plus (ExoQuick) was used according to the manufacturers guidelines. Urinary samples were preprocessed as described above by UC at 13,500 g for 20 min at 4C, filtered through a 0.22 m filter, and concentrated to approximately 5 ml using an Amicon Ultra 10K (for P4) or 100K device. Concentrated urine was thoroughly mixed with 1 ml of ExoQuick-TC Plus reagent and stored at 4C for at least 12 hours. Then, the mixture of the sample and ExoQuick-TC was centrifuged at 1,500 g for 30 min at 4C; the pellet was resuspended in 400 l PBS and stored at -80C for subsequent extraction of RNAs and proteins and for quantification of exosome particles.  Small RNA library construction and sequencing Exosomal RNAs from urine-derived EVs were extracted using miRNeasy serum/plasma kits (Qiagen, Valencia, CA, USA) according to the manufacturers instructions and RNA concentrations were determined by a Quant-IT RiboGreen kit (Invitrogen, Karlsruhe, Germany). RNA size was confirmed using an Agilent RNA 6000 Pico kit and a small RNA kit with an Agilent 2100 Bioanalyzer (Agilent Technologies, Bbligen, Germany). RNA isolated from each sample (10 ng) was used to construct the sequencing libraries with a SMARTer smRNA-Seq kit (Illumina, San Diego, CA, USA) following the manufacturers protocol. Amplified libraries were purified from 6% Novex TBE-PAGE gels (Thermo Fisher, MA, USA) by excision of 138- to 200-bp (18- to 100-bp cDNA plus 120-bp of adaptors) fractions; the resulting cDNA library molecules included sequences on an Illumina flow cell. Libraries were gel-purified and validated by checking the size, purity, and concentration on the Agilent Bioanalyzer. Libraries were quantified using qPCR according to the qPCR quantification protocol guide (KAPA Library Quantification kits for Illumina sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Then, the libraries were pooled in equimolar amounts, and sequencing was performed on an Illumina HiSeq 2500 (Illumina) instrument to generate 101-base reads.   Adapter trimming and identification of known miRNAs Adapter sequences were trimmed using Cutadapt, and the first 3 nucleotides at the 5- and 3-ends of all reads (inserted during the SMART template-switching activity process) were removed. We considered at least 18 base pair size to be reliable after the trimming and used the data for subsequent analysis. We aligned the sequencing reads and quantified miRNA expression by using Bowtie (v1.1.2) and miRDeep2. The human assembly version GRCh37 (hg19) was used and the annotated miRNA information was obtained from miRBase v21.