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| Status |
Public on Jun 12, 2012 |
| Title |
MCF7_H3K4me1 |
| Sample type |
SRA |
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| Source name |
Human breast adenocarcinoma cell-line MCF7
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| Organism |
Homo sapiens |
| Characteristics |
cell-line: MCF7
passage: 5
chip antibody: H3K4me1 (CellSignalingTechnologies, 9723S, lot#1)
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| Growth protocol |
Thawing Cells: 1. Thaw vial immediately in 37º C water bath. Keep O ring above the water surface to prevent contamination. Thaw content with slight shake until only small ice is left in vial. It usually takes 1 min. Spray vial with 70% ethanol all over and wipe its surface with clean tissue in the hood. 2. Open the vial and transfer the content to a 15 ml Falcon tube already containing 5 ml of fresh medium. 3. Spin down at 1000 rpm or 200g for 3 to 5 mins at 4º C. Aspirate supernatant. 4. Resuspend cells in fresh medium and transfer to 150mm x 25mm tissue culture dish. 5. Check the cells under microscope. 6. Cells are cultured in CO2 incubator and medium is changed about every 3 days. 7. It usually takes 3 days or more for cells to recovery from freezing. After cell culture reaches 80-85% confluence, subculture is conducted. Subculture ratio is about 1:3 or 1:4. Passaging Cells: 1. Observe cells to see how confluent they are, whether the cells are alive, whether the cells are contaminated, and whether the cells have the correct morphology. After cell culture reaches 80 to 85% confluence, subculture is conducted 2. Remove media from dish 3. Wash 1x with 10 ml of PBS 4. Add 5 ml of Trypsin and trypsinize for 3 min at 37C. Whack hard – you should see the cells coming down. (Important: never overtrypsinize the cells, so work quickly) 5. Add 5 ml of media and use it to rinse the dish to detach the cells off (4 to 5 times) 6. Spin down at 1000rpm for 3 to 5 min at room temperature. Aspirate supernatant 7. Add 15 ml of media to 15ml tube containing cell pellet, and pipette up and down to mix 8. Add 15 ml of media to each new 150mm x 25mm tissue culture dish (we split 1 dish into 3 to 4 dishes) 9. Add 5mls of cell culturecontaining media to each tissue culture dish. Make sure the media covers the entire area of the dish. Put the cells into 37C with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 10-14 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer 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 |
3 replicates
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using tophat with default parameters
ChIP-seq sequence reads were aligned to the UCSC human genome assembly HG19 using the Eland pipeline (Illumina).
ChIP-seq peaks were called using Solesearch (http://havoc.genomecenter.ucdavis.edu/cgi-bin/chipseq.cgi) with the following settings: 250 bp fragments, alpha-value=0.0001, FDR=0.001, histone modification analysis.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq peak files in gff format, peaks are called from the merged of all replicated samples.
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| Submission date |
Jun 12, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
rui wang |
| E-mail(s) |
ruiw2009@gmail.com
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| Phone |
614-292-6931
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| Fax |
614-688-6600
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| Organization name |
Ohio State University
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| Street address |
212 Biomedical Research Tower,460 West 12th Ave
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| City |
Columbus |
| State/province |
OHIO |
| ZIP/Postal code |
43210 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE38447 |
Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3 |
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| Relations |
| SRA |
SRX153145 |
| BioSample |
SAMN01047940 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM946849_MCF7_H3K4me1_peaks.gff.gz |
278.7 Kb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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