 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 09, 2024 |
| Title |
VCaP, R1881, 10 nM, 24 hours, Input, Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Prostate
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Prostate
cell line: VCaP
cell type: epithelial
chip antibody: Input
compound: R1881
dose: 10 nM
time: 24 hours
|
| Treatment protocol |
LNCaP and VCaP cells were tretaed with either vehicle, or increasing concentrations of low-dose (0.01, 0.03, 0.06, 0.1 nM R1881) or High-Dose (10nM R1881) androgens for 24 hours
|
| Growth protocol |
LNCaP cells (2,000,000 cells/dish) were seeded in 15 cm tissu culture dish in RPMI 1640 supllemented with 8% charcoal dextran treated FBS(CFS). 5 millions cells were collected per condition. For VCaP cell line (5,000,000 cells/dish), cells were seeded in 15 cm tissu culture dish in DMEM (8% CFS). 48 hrs later, cells were treated with R1881 for an additional 24 hrs
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed using a final concentration of 1% methanol-free formaldehyde in PBS at for 10 min and quenched with 125mM glycine for 5 min. Cells were then washed twice in PBS Wash Buffer (Active Motif #53046), pelleted, and stored at -80˚C. For sonication, cells were thawed and resuspended in ice cold Buffer LB1 (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) containing 1x Halt™ 100X Protease and Phosphatase Inhibitor Cocktail (ThermoFisher #78446) on a rotator at 4˚C for 10 minutes. Cells were then centrifuged, liquid decanted, and resuspended in Buffer LB2 (10mM Tris-HCL pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) containing 1x Halt™ 100X Protease and Phosphatase Inhibitor Cocktail. Cells were then centrifuged, liquid decanted and resuspended in ice cold Buffer LB3 (10mM Tris-HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-laurylsarcosine) containing 1x Halt™ 100X Protease and Phosphatase Inhibitor Cocktail. This nuclear resuspension was transferred into Covaris microtube 130 AFA Fiber pre-Slit snap-cap tubes (Covaris #520045) at a maximum concentration of 3 million cells per 130µL and sonicated using the following parameters on a Covaris ME220 according to the manufacturer’s recommendations (Duration (s) = 360, Peak Power = 75, Duty Factor = 5.0, Cycles/Burst = 1000). Following sonication, 10µL of chromatin was removed for analysis of resulting DNA size and concentration. Briefly, 10µL of sonicated chromatin was incubated with 20µg RNase A (Thermo Scientific #EN0531) in TE pH 8 for 30 minutes at 37˚C in a thermal cycler. 20µg of Proteinase K (Thermo Scientific #EO0491) in Active Motif Elution Buffer AM4 (Active Motif #103926) was then added and the sample was incubated for 30 minutes at 55˚C followed by 90 minutes at 65˚C in a thermal cycler. Samples were then cooled to room temperature and DNA was purified using SPRIselect beads (Beckman #B23318) at a bead:sample ratio of 1.4:1 and eluted in 10µL of 10 mM Tris, 0.1 mM EDTA.
Chromatin corresponding to 1 million cells was diluted with 200µL in Active Motif ChIP Buffer and incubated with pre-clearing Protein G agarose beads. After pre-clearing, agarose beads were pelleted and transferred to a new microcentrifuge tube containing 4µg of antibody specific to the target protein (AR: Abcam ab236225). IPs were then incubated overnight on a rotator at 4˚C. Protein G Agarose beads prepared the previous day according to Active Motif’s Low Cell ChIP kit were added to the antibody-bound chromatin and incubated for 4 hours on a rotator at 4˚C. IPs were then transferred to ChIP filtration columns, washed, and eluted in 80µL according to Active Motif’s Low Cell ChIP kit and DNA purified using SPRIselect beads at a bead:sample ratio of 1.4:1. After SPRI cleanup, ChIP samples and reverse crosslinked input DNA was made into sequencing Illumina libraries using Swift NGS 2S Plus (Swift Biosciences #21096) and indexed using Single Indexed Adapters with MIDs (Swift Biosciences #279384). ChIP-seq libraries were prepared using Swift Biosciences Accel-NGS 2S Plus DNA Library Kit with UMIs.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
ChIP-seq reads were trimmed for adaptor sequence/low-quality sequence using Trimmomatic v0.39.
Trimmed ChIP-seq reads were aligned to GRCh38/hg38 using BWA v0.7.17. Reads that were unaligned, multi-mapped, failed platform quality checks or had mapping quality less than 30 were removed.
Peaks were called and narrowPeak files were generated using MACS v2.2.7.1 with --pvalue 0.01, --keep-dup all, --call-summits, and -f BAMPE
BigWig files were generated using bamCoverage v3.5.1 from Deeptools suite, with --normalizeUsing RPGC, --binSize 10, --minFragmentLength 40, --effectiveGenomeSize 2701495761.
Assembly: GRCh38
Supplementary files format and content: bigWig
Supplementary files format and content: narrowPeak
|
| |
|
| Submission date |
Mar 28, 2024 |
| Last update date |
Jul 09, 2024 |
| Contact name |
Xiaodi Qin |
| Organization name |
Duke Cancer Institute
|
| Street address |
2424 Erwin Road
|
| City |
Durham |
| ZIP/Postal code |
27705 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE247593 |
Different doses of androgens induce the expression of distinct gene expression programs |
| GSE262744 |
Different doses of androgens induce the expression of distinct gene expression programs [ChIP-Seq] |
|
| Relations |
| BioSample |
SAMN40647789 |
| SRA |
SRX24093574 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8178015_VCaP_R1881-10_Input_2.sorted.filtered.deduped.bflt.bw |
415.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
