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| Status |
Public on Jan 01, 2016 |
| Title |
HRT1_RNF2_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Hmel human melanocytes, RNF2 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HMEL-BRAFV600E
cell type: immortalized melanocytes
chip antibody: V5 (Abcam ab9116, lot# 754608)
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| Treatment protocol |
Cells were infected with pLenti6.3-RNF2WT or pLenti6.3-GFP lentiviruses, selected with blasticidine (5mg/ml) till control uninfected cells were dead.
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| Growth protocol |
Cells were maintained in standard tissue culture conditions (5% CO2 , 37oC).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells (5 million per antibody) were crosslinked using 1% paraformaldehyde for 10mins at 37 degrees. Reaction was quenched by 0.125M glycine for 5mins, cells washed with PBS and stored at -80oC. Next day cells were thawed on ice and lysed with RIPA buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 140mM NaCl, 1% Triton x-100, 0.2%SDS, 0.1% DOC) for 10min on ice. Sonication was performed using Branson Sonifier 250 to achieve shear length of 200-500bp. Antibodies [Rabbit IgG, V5 (Abcam)] were bound to Dynabeads for 1 hr at 4oC. Extracts were then incubated overnight with antibody-Dynabead mixture. Immune complexes were then washed 5 times with RIPA buffer, once with RIPA-500 (RIPA with 500mM NaCl) and once with LiCl wash buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC). Elution and decrosslinking was performed in direct elution buffer (10mM Tris-Cl pH 8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS) by incubating immune complexes at 65oC for 4-16hrs. Proteinase K (20mg/ml) and RNaseA treatment was performed and DNA cleaned up using SPRI beads (Beckman-Coulter).
Libraries were prepared using NEB reagents as described (Garber et al. 2012). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation, DNA was PCR amplified with Illumina primers for 12 cycles and library fragments of ~300-500 bp (insert plus adaptor and PCR primer sequences) were size selected by SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2000 following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
HMEL-BRAF-RNF2 overexpressing tumor cells
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| Data processing |
Basecalling performed using CASAVA.
RNF2 ChIP-Seq reads were aligned using Bowtie (version 0.12.2) (PMID: 19261174) to human genome assembly NCBI Build 37 (UCSC hg19).
Enriched regions were detected by MACS version 1.4.0 (PMID: 18798982) with a p-value cut-off of 10^-8.
Genome_build: GRCh37
Supplementary_files_format_and_content: Wig files were generated using MACS 1.4.0; Scores represent normalized tag counts for 10 million reads.
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| Submission date |
Oct 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Kadir Caner Akdemir |
| E-mail(s) |
kcakedemir@mdanderson.org
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| Organization name |
MD Anderson Cancer Center
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| Street address |
1515 Holcombe Blvd
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE51929 |
Dual roles of RNF2 in melanoma progression [ChIP-seq] |
| GSE51930 |
Dual roles of RNF2 in melanoma progression |
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| Relations |
| BioSample |
SAMN02419935 |
| SRA |
SRX382354 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1255434_HRT1.wig.gz |
185.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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