|Public on Sep 22, 2023
cell type: NA
sample type: extracellular vesicles (from serum)
treatment: fresh collection
|Prostate tissue slices, epithelial cells, and stromal cells were grown to 70% confluence and then cultured in EV-free media for two days. Evs were isolated from the cell media by differential ultracentifugation.
|Patient-derived prostate epithelial cells were maintained in PrEBM media in humidified atmosphere with 5% CO2 at 37C. Patient-derived prostate stromal cells were maintained in MCDB media with 5% FBS and DHT and Fgfb in humidified atmosphere with 5% CO2 at 37C. Patient-derived prostate tissue slices were maintained in PrEBM media in humidified atmosphere with 5% CO2 at 37C.
|Primary epithelial cell, stromal cell, and tissue slice RNA was harvested using miRNeasy Mini kit (Qiagen). Primary epithelial cell EV, stromal cell EV, tissue slice EV, and serum EV RNA was harvested using the Serum/Plasma miRNeasy kit (Qiagen). 100 ng of total RNA was used for the construction of cell and tissue sequencing libraries. 5 uL of RNA was used for the construction of cell EV, tissue EV, and serum EV sequencing libraries.
microRNA libraries for smallRNA-seq were prepared using the QIAseq miRNA Library Kit following manufacturer's protocols. Libraries were diluted to 10 nM for sequencing.
smallRNA-seq by HiSeq 4000 single read 100 nucleotides
|Illumina HiSeq 4000
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
|GeneGlobe data analysis center
Reads were trimmed using cutadapt, which removed the 3' adapters and low-quality bases
MicroRNA sequences with less than 16 bp and UMI sequences less than 10 bp were removed
MicroRNAs were mapped to miRBase using bowtie, which allowed up to two mismatches
Assembly: miRBase version 22
Supplementary files format and content: Excel files (CPM_tmm_normalizedcounts)
|Mar 23, 2023
|Last update date
|Sep 22, 2023
|University of Illinois at Chicago College of Medicine
|909 South Wolcott Ave
|Prostate tissue explant, epithelial cell, stromal cell, and extracellular vesicle microRNA characterization by NGS