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Sample GSM5004273

Query DataSets for GSM5004273

Status

Public on Mar 01, 2021

Title

MV34_C.IonXpressRNA_010

Sample type

SRA

Source name

Control cells rep 4

Organism

Homo sapiens

Characteristics

cell type: MV3 melanoma cells
genotype/variation: transfection with empty pcDNA.3 plasmid

Treatment protocol

The day prior to transfection, cells were seeded at a density of 10 500 cells/cm2. To achieve transient overexpression of MCPIP1-wt and MCPIP1-D141N pcDNA3 plasmid vecor with the expression cassette for the respective MCPIP1 form was used. As the corresponding control transfection with empty vector was conducted. For transfection, 3 µg of DNA per well of a 6-well plate was used. The DNA was mixed with polyethyleneimine (PEI; 408727, Sigma, Darmstadt, Germany) at a ratio of 1:3 and diluted in OPTI-MEM (11058021, Gibco, Waltham, MA, USA). The cells were incubated with the DNA:PEI solutions for 6 hours. Twenty-four hours following transfection, the medium was changed to selection medium containing 900 µg/ml geneticin (G418, Lab Empire, Rzeszów, Poland). The selection medium was changed daily until the 4th day after transfection, when the cells were collected.

Growth protocol

MV3 melanoma cells were cultured in RPMI 1640 medium (R8758, Sigma, Darmstadt, Germany) supplemented with 10% foetal bovine serum (10270, Gibco, Waltham, MA USA), 1% non-essential amino acids (M7145, Sigma, Darmstadt, Germany) and 50 μg/ml gentamycin (G1397, Sigma, Darmstadt, Germany) at 37°C in an atmosphere containing 5% CO2. Cells were routinely assessed for mycoplasma contamination. All tests were negative.

Extracted molecule

total RNA

Extraction protocol

RNA was isolated using the miRVana™ miRNA Isolation Kit (AM1560, Thermo Fisher Scientific, Waltham, MA, USA).
Libraries were generated with an Ion Total RNA-Seq Kit v2 (4479789, Thermo Fisher Scientific, Waltham, MA, USA).
As the input material, 10 µg of total RNA per sample was used.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Ion Torrent Proton

Data processing

verification of the quality of the reads (FastQC software version 0.11.5 )
trimming of adapters of raw sequencing reads (Cutadapt version 2.08 )
reads alignment (Bowtie version 1.2.3)
Read normalization and identification of differentially expressed miRNAs (DESeq2 version 1.24.0 )
Genome_build: miRBase
Supplementary_files_format_and_content: excel, list of differentially expressed miRNAs for wt vs c, d141n vs c and overall

Submission date

Jan 04, 2021

Last update date

Mar 01, 2021

Contact name

Iwona Nowak

E-mail(s)

[email protected]

Phone

694094603

Organization name

Jagiellonian University