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| Status |
Public on Aug 03, 2023 |
| Title |
LacZ - D2d - AR |
| Sample type |
SRA |
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| Source name |
A375 melanoma cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A375
treatment: Dabrafenib
transfection: control
chip antibody: 5ug of anti-Androgen Receptor, PG-21, Cat. No. 06-680 (Sigma)
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| Treatment protocol |
A375 melanoma cells were treated with Dabrafenib (0.5uM) versus vehicle (DMSO) control for 48h.
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| Growth protocol |
All melanoma cell lines and patient-derived primary melanoma cells were maintained in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde for 10 minutes at RT, followed by quenching with glycine (final concentration 125 mM). After washing with ice-cold PBS, cells were collected through centrifugation (400g). For the analysis of total lamin A/C chromatin binding, cells were lysed and chromatin was prepared using the Chromatin EasyShear Kit - High SDS (Diagenode; Cat# C01020012), and sonicated using an E220 focused ultrasonicator (Covaris). The ChIP reaction was performed using the iDeal ChIP-seq kit (Diagenode, Cat# C01010055). Samples were pre-cleared using the beads provided in the kit and incubated overnight at 4°C with 5ug of anti-Androgen Receptor, PG-21, Cat. No. 06-680 (Sigma). Antibody-chromatin complexes were pulled down using protein A-coated magnetic beads. Elution was performed as per the manufacturer's instructions and chromatin was quantified using the Qubit Fluorometric Quantification Kit (Thermo Scientific). For library preparation, 10 ng of DNA was utilized with the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina. Subsequently, sequencing was performed at the Novegene facility using the Novaseq 6000 platform.
For library preparation, 10 ng of DNA was utilized with the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Data analysis was carried out using the web tool https://usegalaxy.org/. Briefly, Bowtie2 [http://bowtie-bio.sourceforge.net/] was used for fastq files alignments and, for peak detection, MACS software [http://liulab.dfci.harvard.edu/MACS/] with default parameters. The Integrative Genomics Viewer (IGV) [http://www.broadinstitute.org/igv] was used for a graphic illustration of ChIP-Seq peaks and ENCODE data (http://genome.ucsc.edu/ENCODE/) for information on chromatin organization. Raw data files from ChIP-seq assays were aligned to the GRCh38 reference genome with Bowtie2. MACS2 software was used for peak detection, with a q-value cutoff of 0.05. Peaks were annotated and merged with the annotatePeaks.pl and mergepeaks.pl functions, available within the HOMER software.
Assembly: GRCh38
Supplementary files format and content: BigWig files
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| Submission date |
May 17, 2023 |
| Last update date |
Aug 03, 2023 |
| Contact name |
Paola Ostano |
| Organization name |
Fondazione Edo ed Elvo Tempia
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| Street address |
via Malta 3
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| City |
Biella |
| ZIP/Postal code |
13900 |
| Country |
Italy |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE232690 |
Androgen Receptor is a Determinant of Melanoma BRAFi/MEKi Resistance [ChIP-Seq] |
| GSE232697 |
Androgen Receptor is a Determinant of Melanoma BRAFi/MEKi Resistance |
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| Relations |
| BioSample |
SAMN35130956 |
| SRA |
SRX20415427 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7371844_LacZ_D2d_AR.bw |
57.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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