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

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

从3名不同供体获取的原代正常人表皮黑色素细胞（Normal Human Epidermal Melanocytes, NHEM）以及3株黑色素瘤细胞系（WM35、WM9与WM902B）均进行了短时培养（培养时长为48小时）。随后通过包括超速离心在内的多步离心法，从条件培养基（Conditioned Medium, CM）中分离得到细胞外囊泡（Extracellular Vesicles, EVs）。为探究细胞外囊泡中的miRNA载荷以及对应细胞内的miRNA表达谱，我们提取了总RNA。取10~50 ng总RNA，按照制造商说明书，使用NEXTflex Small RNA-seq Kit v3（Bioo Scientific）完成小RNA建库流程。将混合后的文库以10 nM的浓度用于测序。按照仪器制造商说明书，使用Illumina NextSeq 550测序仪在莱比锡大学医学院莱比锡临床研究中心（IZKF Leipzig）测序核心实验室完成1×75 bp的单端测序。使用带有文库插入片段旁侧随机碱基的NEXTflex试剂盒接头序列，对原始测序reads进行拆分、接头修剪与质量过滤。使用Bowtie2工具将过滤后的reads比对至人类参考基因组（hg38）以及miRbase参考序列（v22）。使用R语言Bioconductor工具包Rsamtools（http://bioconductor.org/packages/release/bioc/html/Rsamtools.html）计算reads计数，并通过DESeq2与EdgeR这两款R语言Bioconductor工具包完成数据标准化。注：wc 指代全细胞；exo 指代细胞外囊泡。