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| Status |
Public on Sep 05, 2017 |
| Title |
LNCaP_NOMe_4hr_1 |
| Sample type |
SRA |
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| Source name |
Human cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell type: LNCaP clone FGC (ATCC CRL-1740). Androgen-sensitive human prostate adenocarcinoma cells
passage number: 72-73
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| Treatment protocol |
Human LNCaP prostate cancer cells were maintained in RPMI Medium 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), for at least 72 hrs to 70-80% confluence. LNCaP cell were then seeded to 2.5x106 cell per 15cm dish and starved for 3 days in RPMI Medium 1640-no-phenol red (Gibco) and 5% chracoal stripped FBS (CSFBS). Then LNCaP cells were treated with or without (vehicle, ETOH) 10nM of DHT for 2h, 4h and 16h. Cells were harvested for nuclei extraction.
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| Growth protocol |
PrEC cells were growth and maintained in PrEGM media (Cambrex) at 37ºC with 5% CO2; LNCaP cells were growth and maintained in T-medium (Gibco) and 10% fetal bovine serum (FBS) at 37ºC with 5% CO2. When reached 70-80% confluence cell were harvested for nuclei extraction.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized and centrifuged for 7 mins at 1000rpm, then washed in ice-cold PBS and resuspended in 1mL ice-cold Nuclei Buffer (10mM Tris,pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA and 1% IGEPAL, plus protease inhibitors) per 5x106 cells and incubated on ice for 10 min. To obtain the nuclei, cells were dounce-homogenized for 15 times and incubated on ice for about 45 min, until single nuclei were visualized under the microscope. Nuclei were recovered by centrifugation at 3000 rpm for 5 min and washed in Nuclei Wash Buffer (10mM Tris,pH 7.4, 10mM NaCl, 3mM MgCl2 and 0.1mM EDTA containing protease inhibitors). Freshly prepared nuclei (2x105 cells) were resuspended in 1X M.CviPI reaction buffer (NEB), then treated with 150U of M.CviPI (NEB; 50,000U/mL) in 15µL 10X reaction buffer, 45µL 1M sucrose and 0.75µL SAM in a volume of 150uL. Reactions were quenched by the addition of an equal volume of Stop Solution (20mM Tris-HCl [pH 7.9], 600mM NaCl, 1% SDS, 10mM EDTA, 400µg/ml Proteinase K) and incubated at 55°C overnight. DNA was purified by phenol/chloroform extraction and ethanol precipitation.
50ng of genomic DNA was bisulfite converted using the EZ DNA Methylation Gold kit (Zymo Research, Cat: D5005) following the manufacturer’s protocol. The bisulfite converted DNA was then subjected to library preparation using the EpiCenter kit (now known as the EpiGnome library preparation kit or TruSeq DNA Methylation kit, Illumina, Cat: EGMK81312). In this “post bisulfite conversion” library preparation method, single stranded bisulfite converted DNA was randomly primed to synthesize DNA strands with a specific sequence tag followed by 3’ tagging and PCR amplification using the FailSafe PCR enzyme (Gene Target Solutions,Cat: FSE51100). The PCR was performed for 10 cycles followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Cat: A63881). The purified library was eluted in a final volume of 20ul pure water. Quality of the library obtained was checked using the DNA High sensitivity chip on the Agilent Bioanalyzer. The library was quantified using Qubit and KAPA Biosystems Library quantification kit according to manufacturer’s instructions.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw read pairs were aligned to the human genome (hg19) using bwa-meth, a bisulfite aware wrapper for the bwa-mem aligner.
Read pairs with identical strand, start and end positions were considered PCR duplicates and removed from downstream analysis using MarkDuplicates from the Picard toolset.
BisSNP v0.82.2 was then used to extract the methylation status of each WCG and GCH site in each sample which was then transformed into a "bigTable" of counts of methylation and coverage.
Nucleosome deplete regions (NDRs) were detected using the findNDRs function of the aaRon R package https://github.com/astatham/aaRon
NDRs were detected by a 100bp sliding window in 20bp increments chi-squared test of GCH methylation versus the genome background with a p-value cutoff of -log10(p)>15,
significant windows overlapped and retained if a minimum 140bp in size.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph: NDR bed files contain the -log10 pvalue in the name field (4th column)
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| Submission date |
Dec 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Fatima Valdes Mora |
| E-mail(s) |
f.valdesmora@garvan.org.au
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| Phone |
+61 2 92958334
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| Organization name |
Garvan Institute of Medical Research
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| Department |
Genomics and Epigenetics
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| Lab |
Histone Variant Group
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| Street address |
384 Victoria Street
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| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE76334 |
Novel contribution of acetylated histone variant H2A.Z in activation of neo-enhancers in prostate cancer [NOMe-seq] |
| GSE76337 |
Novel contribution of acetylated histone variant H2A.Z in activation of neo-enhancers in prostate cancer |
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| Relations |
| BioSample |
SAMN04370250 |
| SRA |
SRX1502202 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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