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| Status |
Public on Aug 21, 2019 |
| Title |
HFD_WT_PHF8 |
| Sample type |
SRA |
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| Source name |
WT ventral prostates
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| Organism |
Mus musculus |
| Characteristics |
cell line: ventral prostatic tissues
genotype/variation: wild type
antibody: anti-PHF8
antibody manufacturer: Bethyl Laboratories
antibody catalog number: #A301-772A
diet: HFD
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| Growth protocol |
FVB Hi-MYC mice, expressing the human c-MYC transgene in prostatic epithelium, were obtained from the National Cancer Institute Mouse Repository at Frederick National Laboratory for Cancer Research5. Upon weaning, male mice heterozygous for the transgene (MYC), together with their wild type littermates (WT), were fed a purified control diet (CTD; Harlan Laboratories, TD.130838) consisting of 10% fat, or a high-fat diet (HFD; Harlan Laboratories, TD. 06414) consisting of 60% fat; ingredients were adjusted on a kcal basis. Food was changed on a weekly basis, and mice were weighed every three weeks, starting at weaning. Animals were kept on a 12-hour light / 12-hour dark cycle, and allowed free access to food and water at the Dana-Farber Cancer Institute (DFCI) Animal Resources Facility. The animal protocol was reviewed and approved by the DFCI Institutial Care and Use Committee (IACUC), and was in accordance with the Animal Welfare Act.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fresh-frozen VP tissues from 12-week-old mice were pulverized (Cryoprep Impactor, Covaris), resuspended in PBS + 1% formaldehyde, and incubated at room temperature for 20 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration) for 15 minutes at room temperature, then washing in ice cold PBS + EDTA-free protease inhibitor cocktail (PIC; #04693132001, Roche). Multiple biological replicates were combined for each condition in two distinct pools (replicates). Chromatin was isolated by the addition of lysis buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0), 0.1% NaDOC, 0.13 M NaCl, 1X PIC) + sonication buffer (0.25% sarkosyl, 1 mM DTT) to the samples, which were maintained on ice for 30 minutes. Lysates were sonicated (E210 Focused-ultrasonicator, Covaris) and the DNA was sheared to an average length of ~200-500 bp. Genomic DNA (input) was isolated by treating sheared chromatin samples with RNase (30 minutes at 37°C), proteinase K (30 minutes at 55°C), de-crosslinking buffer (1% SDS, 100 mM NaHCO3 (final concentration), 6-16 hours at 65°C), followed by purification (#28008, Qiagen). DNA was quantified on a NanoDrop spectrophotometer, using the Quant-iT High-Sensitivity dsDNA Assay Kit (#Q33120, Thermo Fisher Scientific). On ice, ChIP-validated H4K20me1 (2 micrograms, #ab9051, Abcam) or PHF8 (5 micrograms, #A301-772A, Bethyl Laboratories) antibodies were conjugated to a mix of washed Dynalbeads protein A and G (Thermo Fisher Scientific), and incubated on a rotator (overnight at 4°C) with 1.5 micrograms (H4K20me1) or 5 micrograms (PHF8) of chromatin. ChIP’ed complexes were washed, sequentially treated with RNase (30 minutes at 37°C), proteinase K (30 minutes at 55°C), de-crosslinking buffer (1% SDS, 100 mM NaHCO3 (final concentration), 6-16 hours at 65°C), and purified (#28008, Qiagen). The concentration and size distribution of the immunoprecipitated DNA was measured using the Bioanalyzer High Sensitivity DNA kit (#5067-4626, Agilent).
Dana-Farber Cancer Institute Molecular Biology Core Facilities prepared libraries from 2 ng of DNA, using the ThruPLEX DNA-seq kit (#R400427, Rubicon Genomics), according to the manufacturer’s protocol; finished libraries were quantified by the Qubit dsDNA High-Sensitivity Assay Kit (#32854, Thermo Fisher Scientific), by an Agilent TapeStation 2200 system using D1000 ScreenTape (# 5067-5582, Agilent), and by RT-qPCR using the KAPA library quantification kit (# KK4835, Kapa Biosystems), according to the manufacturers’ protocols; ChIP-seq libraries were uniquely indexed in equimolar ratios, and sequenced to a target depth of 40M reads on an Illumina NextSeq500 run, with single-end 75bp reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
12-week-old FVB
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| Data processing |
For all samples reads were aligned to their indicated build using bowtie2 with parameters -k 1
Bam alignments were converted to bedgraph using bedtools genomecoverage and scaled to million mapped reads
Bigwig files were generated from scaled bedgraphs using UCSC bedGraphTobigWig tool
Genome_build: mm9
Supplementary_files_format_and_content: Processed data are in bigwig format and contain whole genome coverage
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| Submission date |
Dec 05, 2016 |
| Last update date |
Aug 21, 2019 |
| Contact name |
James Bradner |
| E-mail(s) |
[email protected]
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Medical Oncology
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| Lab |
Bradner Lab
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| Street address |
450 Brookline
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE90910 |
High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program |
| GSE90912 |
High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program |
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| Relations |
| BioSample |
SAMN06111849 |
| SRA |
SRX2396659 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2417447_Merge_Pool_5_6_HFD_WT_PHF8.bigwig |
626.1 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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