Project description:RI-SEC-seq: small RNA sequencing in size-exclusion chromatography fractions of MCF-7 cell-conditioned medium treated with/without RNase inhibitor
Project description:The current study is focused on Isolating EVs and their cargo content (miRNAs) from culture medium conditioned by individual embryos that can differentiate between the blastocyst and non-blastocyst embryos. We were able to isolate EVs from both blastocyst and non-blastocyst group with size exclusion chromatography (Izon’s™ qEV single column) and characterize them with Western blotting, nanoparticle tracking analysis, and Transmission electron microscopy. Further, we could isolate total RNA (including small RNAs) from the blastocyst and non-blastocyst EVs for miRNA sequencing.
Project description:Plasma was harvested from two cohorts of facility-matched germ free (GF) and specific-pathogen free (SPF) mice at the National Gnotobiotic Rodent Resource Center (NGRRC; North Carolina, USA). Plasma was then fractionated by size-exclusion chromatography using three tandem Superdex200 increase columns (Cytiva). Fractions corresponding with HDL were then pooled and concentrated prior to RNA isolation. Small RNA libraries were generated from total RNA using NextFlex V3 Small RNA Seq-kit (Perkin Elmer) according to manufacturer’s instructions. Equimolar amounts of each library were then pooled and sequenced on the NextSeq500 platform (Illumina). Individual libraries were then demultiplexed and analyzed with the TIGER analytical pipeline.
Project description:To evaluate performance of immunomagnetic separation and size exclusion chromatography in the isolation of different extracellular RNA carriers from biofluids.
Project description:Plasma from lung cancer patients from EDTA tubes was fractionated using size exclusion chromatography. Fractions 1-5, 7-11, 12-15, 16-20 were pooled, cfDNA was extracted from the fractions and paired unfractionated samples and PE150bp sequencing was performed on an Illumina Novaseq S4 flowcell. Samples are provided as raw reads without any prior processing.
Project description:Paracrine signals relayed between heterogenous cell types through small and large extracellular vesicles promote cancer cell growth, invasion and metastasis. Microplasts, also called as cytoplasts or cytokineplasts are large extracellular vesicles (0.5-11µm diameter) originating from migratory cells. Microplasts are known to be associated with invasive phenotypes of cancer cells and promote metastasis. Treatment with macrophage conditioned medium induces significant shedding of microplasts from MCF-7 breast adenocarcinoma cells. To characterise microplasts and to study their potential role in intercellular communication in cancer, we isolated microplasts derived from macrophage conditioned medium treated MCF-7 cells and investigated their protein cargo through LC-MSMS.
Project description:We studied extracellular vesicles (EVs) from synovial fibroblast (SF). EVs were isolated from the secretome of non-senescent and irradiation-induced senescent SFs using size exclusion chromatography. EV RNA was extracted and subject to small RNA sequencing on an Illumina NovaSeq SP using 100bp, single end reads. After mapping against GRCh38.p12 and miRBase v22.1 differential expression analysis was undertaken with edgeRv3.28 using a quasi-likelihood negative binomial generalized log-linear model. We identified a panel of differentially expressed small non-coding RNAs.
Project description:Experiment aimed at understanding the compositional changes of small extracellular vesicles (sEVs) derived from cells grown under hyperthermic conditions. Human embryonic kidney (HEK293) cells were cultured 48 h under normal conditions (37°C/140 rpm/5% CO2) before being subjected to a 72 h, 40°C temperature shift. sEVs were then isolated from the supernatant via tangential flow filtration (TFF) and size exclusion chromatography (SEC)
Project description:We performed quantitative proteomics analysis by Stable Isotope Labelling by Amino Acids in Cell culture (SILAC) to understand how centrosome amplification changes the composition of human small extracellular vesicles. We carried out SILAC labelling with medium and heavy isotopes, as it enables the exclusion of contaminant serum proteins, which would be unlabeled (equivalent of light labeling), as well as allowing for simultaneous processing of purification steps to decrease sample-to-sample variability. We isolated small extracellular vesicles by ultracentifugation followed by size exclusion chromatography (SEC). All experiments were performed in duplicate with switched SILAC labelling.
Project description:Small RNA sequencing of human and macaque brain tissue and brain-derived extracellular vesicles separated by size exclusion chromatography