NGS analyses of miRNA cargo in extracellular vesicles from melanoma cell lines and normal melanocytes as well as the endogenous miRNA expression of these cells

Published: 19 November 2021| Version 1 | DOI: 10.17632/g9b8k73vzx.1
Contributor:
Dennis Gerloff

Description

Primary normal human epidermal melanocytes (NHEM) from three different donors and three melanoma cell lines (WM35, WM9 and WM902B) were briefly cultured (48 h). EVs were isolated from the conditioned medium (CM) by different centrifugation steps, in-cluding ultracentrifugation. To investigate the miRNA cargo in EVs and the miRNA expression in the corre-sponding cells, total RNA was isolated. 10-50 ng of total RNA was used in the small RNA protocol with the NEXTflex Small RNA-seq Kit v3 (Bioo Scientific) according to the instructions of the manufacturer. A pool of libraries was used for sequencing at a concentration of 10 nM. Sequencing of 1x75 bp was performed with an Illumina NextSeq 550 sequencer at the sequencing core facility of the IZKF Leipzig (Faculty of Medicine, University Leipzig) according to the instructions of the manufacturer. Demultiplexing of raw reads, adapter trimming and quality filtering was done, using the adapter sequences of the NEXTflex kit containing random bases next to the library insert. Mapping against the human reference genome (hg38) and miRbase reference sequences (v22) was done using Bowtie2 . Read counts were calculated with the R bioconductor package Rsamtools (http://bioconductor.org/packages/release/bioc/html/Rsamtools.html) and normalised us-ing the DESeq2 and EdgeR R bioconductor packages. wc: whole cells; exo: extracellular vesicles

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Human Cell, Melanoma

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