|
| Status |
Public on Jul 01, 2024 |
| Title |
MITF bio rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
A2058_MITF
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A2058
cell type: human melanoma cell line
treatment: LVX MITF vector
|
| Treatment protocol |
Briefly, lentivirus was generated using Lenti-X 293 HEK cells. Two mastermixes were prepared: (1) 557μl reaction buffer (Clontech), 36μl (Clontech) and 7μg of pLVX vector-containing plasmid of interest; (2) 592.5μlreaction buffer (Clonetech) and 7.5μl polymer (Clontech). The two mastermixes were combined and incubated at room temperature for 20mins. The combined mastermix was added to the 10cm dish as droplets and incubated overnight at 37°C. The virus containing media was replaced by normal growth medium and incubated at 37°C for a further 48hrs. Virus particles were harvested by passing the media through a 0.45μm filter and were stored in -80°C until used. Lentiviral transduction was performed on cells grown in a 6-well plate at 70% confluency in media containing 8μg/ml polybrene. Viral particles were added into wells and centrifuged at room temperature at 2830rpm for 1 hr 30 minutes. media was replaced 24 hours post-centrifugation. Cells were then selected in media containing 1μg/μl puromycin prior to performing experiments. siRNA transfection was performed using Lipofectamine 2000 as per the manufacturers protocol, with an siRNA concentration of 50 mM for 48 hours.
|
| Growth protocol |
Cells were seeded into 10cm dishes and cultured in DMEM at 70% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Overexpression and knockdown were confirmed using western blot prior to RNA isolation. Once validated, RNA was extracted from these cells using an RNA extraction kit from Qiagen. Sample quality was confirmed using a Nanodrop and QC analysis was performed using a bioanalyzer to obtain RIN values. Samples with appropriate RIN values were then chosen such that we had three independent triplicate samples for each condition.
RNA library preparation was performed using a Tru Seq stranded total RNA kit from Illumina. The library pool was diluted and denatured according to the standard NextSeq protocol (Document # 15048776 v02).
Sequencing was performed using the Illumina NextSeq 500 (NextSeq control software v2.1.0 / Real Time Analysis v2.4.11) to generate paired-end 76 bp reads using a 150 cycle NextSeq500/550 High Output reagent Kit v2 (illumina, FC-404-2002). Sequencing was performed at the Institute for Molecular Bioscience Sequencing Facility (University of Queensland).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Paired-end bulk RNA-seq of A2058 melanoma cells treated with an LVX MITF vector.
|
| Data processing |
Raw sequencing data was converted to fastq files using bcl2fastq2 v2.18.0.
Paired-end bulk RNA-seq fastq file reads were mapped to the Hg19 reference genome using STAR v2.6.0.a with parameters “--outFilterMultimapNmax 2 --outSAMtype BAM Unsorted”.
Bam files were then sorted using samtools v1.10 with default settings
Gene counts were quantified using featureCounts v1.6.3 with parameters “-p -t exon”.
Genome_build: Hg19
Supplementary_files_format_and_content: counts.txt.gz, sample_meta.txt
|
| |
|
| Submission date |
Feb 25, 2021 |
| Last update date |
Jul 01, 2024 |
| Contact name |
Brad Balderson |
| E-mail(s) |
[email protected]
|
| Organization name |
The University of Queensland
|
| Department |
The School of Chemistry and Molecular Bioscience
|
| Lab |
Boden Lab, Thor Lab
|
| Street address |
Chemistry Bld, 68 Cooper Rd
|
| City |
Brisbane |
| State/province |
QLD |
| ZIP/Postal code |
4072 |
| Country |
Australia |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE167496 |
MITF perturbation in A2058 Melanoma cell lines |
|
| Relations |
| BioSample |
SAMN18056009 |
| SRA |
SRX10167836 |
