Niversity, Shinjyuku-ku, Japan; dDepartment of Molecular and Cellular Medicine, Institute of Health-related Science, Tokyo Medical University, Shinjyuku-ku, Japan; eHamamatsu University School of Medicine, Hamamatsu, JapanOT09.Stringent tiny extracellular vesicle purification and ligationindependent compact RNA-seq: new insights into released RNA populations Kenneth W. Witwera, Tine Sch ena, Yiyao Huanga, Andrey Turchinovichb, Senquan Liua, Linzhao RGS19 Molecular Weight Chenga and Vasiliki MachairakicaJohns Hopkins University College of Medicine, Baltimore, USA; bSciBerg, Heidelberg, Germany; cJohns Hopkins University, Baltimore, USAIntroduction: Small extracellular vesicles (sEVs) are nanometre-sized vesicles secreted from numerous cell types. Exosomes, a variety of sEVs, derived from multivesicular bodies (MVBs), mediate cell-to-cell communication by transporting proteins, mRNAsand miRNAs. The delivery of proteins between cells by sEVs, such as exosomes, is associated with tumour progression and neurodegenerative diseases. However, the molecular mechanism by which proteins are sorted to sEVs will not be totally understood. Approaches: By utilizing immunoprecipitation, immunocytochemical, electron microscopic and proteomics evaluation, we report that ubiquitin-like three (UBL3)/ membrane-anchored Ub-fold protein (MUB), an evolutionarily conserved protein, acts as a novel posttranslational modification (PTM) aspect that regulates protein sorting to sEVs. Results: We locate that UBL3 modification is by way of cysteine residues only under non-reducing conditions and is indispensable for sorting of UBL3 to MVBs and sEVs. In addition, we observe a 60 reduction of total protein, but not RNA, levels in serum sEVs purified from UBL3-knockout (KO) mice compared withIntroduction: MicroRNAs are a significant concentrate of exRNA and EV studies. Lots of publications report miRNAs as the plurality or majority of released small RNAs. On the other hand, TrkA Gene ID legacy sRNA profiling strategies are biased towards miRNAs. Abundant RNAs outdoors vesicles also contaminate lots of EV preparations. We sequenced exRNA from induced pluripotent stem cells (iPSCs) having a ligation-independent strategy: ultra-low-input capture and amplification by tailing and sequencing (CATS). Procedures: Culture conditioned medium (CCM) was collected from four lines of count-normalized iPSCs over three passages ( 200 mL/passage). Fractions had been: cells (washed/lysed); “whole releasate” = clarified CCM (300 x g, 2k x g); “large EVs (lEVs)” = pellet of 10k x g spin; “small EVs (sEVs) = preparation by tangential flow filtration (100 kDa cutoff) and size exclusion chromatography (Izon); and “soluble” = flow-through from sEV preparation. Particles have been counted by ParticleMetrix, visualized by TEM, and tested for up to 7 positive or damaging markers per MISEV2014/18. lEVs and sEVs were treated with nucleases. CATS sRNA libraries had been analysed for contribution ofISEV2019 ABSTRACT BOOKRNA classes. Statistics were corrected for several comparisons; significance = corrected p 0.01. Benefits: Making use of CATS, miRNAs mapped at only a modest of total sRNA reads; generally significantly less than 1 . Nucleasetreated sEVs had drastically lower relative miRNA levels than cells or soluble releasate. tRNAs/fragments had highest relative abundance in entire releasate and soluble fractions, albeit with substantial variability. Significantly distinctive in most releasate fractions vs cells have been sno/scaRNA, mRNA, and lncRNA. Cellular distribution differed only from lEV and sEV for RNU RNAs, and only from sEV for Y RNAs. rRNAs/f.