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| Status |
Public on Aug 20, 2019 |
| Title |
MED1-chip_LNCaP_DHT |
| Sample type |
SRA |
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| Source name |
LNCaP
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Prostate Cancer
cell line: LNCaP
treatment: AR stimulation (6h)
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| Treatment protocol |
AR stimulation: Cells were plated in complete media at the required confluency. After 24h cells were washed with DPBS and cultured in RPMI/DMEM without Phenol Red (Invitrogen, 11835030) and 10% charcoal-dextran stripped FBS (Gemini Bio-Products, 100-119) for 72 h. Cells were then stimulated with 10 nM DHT for required time points.
THZ1 and enzalutamide treatment: In DHT starvation and stimulation experiments, cells were treated with CDK7 inhibitor-THZ1 (MedChem Express, HY-80013A/CS-3168) or Enzalutamide (Selleckchem, S1250) 30-45 min before DHT stimulation. Otherwise, the cells were treated with the particular drugs/compounds 24h post seeding. Enzalutamide and DHT was dissolved and aliquoted in DMSO and methanol respectivily.
MED1 and CDK7 knockdown protocol: For knockdown experiments, cells were seeded in six-well plates and transfected with 100 nM ON-TARGETplus SMARTpool siRNA (Dharmacon) targeting MED1 (Dharmacon, L-004126-00-0005), CDK7 (Dharmacon, L-003241-00-005), and non-targeting Pool as a control (Dharmacon, D-001810-10-05) using lipofectamine RNAiMAX (Invitrogen,13778150) according to the manufacturer's instructions. Cells were then harvested 48/72h post-transfection and used in RNA-seq and other experiments.
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| Growth protocol |
LNCaP, and DU145 prostate cancer cell lines were grown in RPMI 1640 (Gibco, 11875093), and VCaP prostate cancer cell line was grown in DMEM with Glutamax (Gibco, 21013024). The medium was supplemented with 10% of FBS (HYC, SH30910.03) and 1% of Penicillin Streptomycin Solution (Invitrogen, 15140122).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
VCaP cells were serum starved, for 72h followed by 12h treatment with 200 nM THZ1 in presence/absence 10 nM DHT. ChIP was performed using iDeal ChIP-seq Kit for Transcription Factors (Diagenode, C01010170) according to manufacturer's protocol. In brief, the cells were crosslinked with 1% formaldehyde in culture medium for 10 min at room temperature. Cross-linking was terminated by the addition of 1/10 volume 1.25 M glycine for 5 min at room temperature followed by cell lysis and sonication (Bioruptor, Diagenode), resulting in an average chromatin fragment size of 200 bp. Chromatin equivalent to 5×106 cells was isolated and incubated with 10 micrograms antibody overnight at 4 °C (MED1, and AR) (Diagenode). ChIP-seq libraries were prepared from the ChIP-enriched DNA samples using the TruSeq ChIP Library Preparation Kit (Illumina, IP-202-1012, IP-202-1024) and protocol. In brief, ChIP-enriched DNA (1-10 ng) was converted to blunt-ended fragments. A single A-base was added to fragment ends followed by ligation of Illumina adaptors. The adaptor-modified DNA fragments were enriched by PCR using the Illumina Barcode primers and Phusion DNA polymerase. PCR products were size selected using 3% NuSieve agarose gels (Lonza) followed by gel extraction using QIAEX II reagents (QIAGEN). Libraries were quantified with the Bioanalyzer 2100 (Agilent) and sequenced on the Illumina NextSeq 500 Sequencer (75 nucleotide read length).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
MED1 ChIP DNA
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| Data processing |
Illumina Nextseq500 reads were demultiplexed using bcl2fastq
Read quality was checked for each sample usign FastQC.
Alignment of reads were performed using STAR aligner with the default setting in Illumina Basespace (https:// basespace.illumina.com/home/indexIllumina)
For ChIP-seq data, the bam files were converted to bigwig tracks using bamcoverage from deeptools
For ChIP-seq data, enrichment peaks were computed using MACS2
Genome_build: hg19
Supplementary_files_format_and_content: bigwig
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| Submission date |
Jan 17, 2019 |
| Last update date |
Aug 20, 2019 |
| Contact name |
Irfan Asangani |
| E-mail(s) |
asangani@upenn.edu
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| Organization name |
University of Pennsylvania
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| Department |
Cancer Biology
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| Street address |
421 Curie Bld.
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| City |
PHILADELPHIA |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE125245 |
CDK7 inhibition suppresses Castration-Resistant Prostate Cancer through MED1 inactivation |
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| Relations |
| BioSample |
SAMN10764516 |
| SRA |
SRX5258172 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3567216_MED1-chip_LNCaP_DHT.bw |
131.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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