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| Status |
Public on Feb 21, 2012 |
| Title |
RNA Pol II Flavo 60min (reanalysis) |
| Sample type |
SRA |
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| Source name |
RNA Pol II Flavo 60min
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
chip antibody: N-20 antibody (Santa Cruz, sc-899)
treatment: 60min flavopiridol treatment
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| Growth protocol |
HeLa cells were maintained at 5% CO2 and 37°C in DMEM plus 10% FBS (Hyclone). Cells were grown in T-150 flasks to 90% confluence and treated for 5 or 60 minutes with Flavopiridol (final concentration 1 μM with 0.1% DMSO) or 0.1% DMSO alone for control.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each immunoprecipitation, 5 x 10^7-1 x 10^8 cells were used. 1/10 volume of fresh 11% paraformaldehyde solution was added directly into the plates and cells were cross-linked for 15 minutes at room temperature. Cross-linking was stopped by adding glycine. Cells were washed and pelleted in PBS and resuspended in ice cold Lysis Buffer 1 for 10 minutes, then pelleted and resuspended in ice cold Lysis buffer 2 for another 10 minutes, and then the cells were pelleted and resuspended in ice cold Lysis buffer 3. After sonication, DNA fragments (<500 bp) were spun to pellet debris. For each immunoprecipitation 100 μL of Protein G Dynabeads (Invitrogen) were used. The beads were washed with Block Solution three times and resuspended in 250 μL Block Solution. The beads were incubated with 10 μg of RNAP II antibody at 4°C overnight. For ChIP-Seq, the N-20 antibody (Santa Cruz, sc-899) was used which recognizes the N-terminus of Rpb1. Then the beads were washed with Block Solution three times, resuspended in 100 μL of Block Solution, and incubated with the sonicated cell lysate at 4°C overnight. After the incubation, beads were washed four times with RIPA buffer, once with a buffer containing TE and 50 mM NaCl. Immunocomplexes were eluted for 30 minutes at 65°C with Elution buffer and the beads were removed with a magnetic concentrator. Reverse crosslinking was performed for both the immunoprecipitated DNA and for the input DNA samples by an incubation at 65°C for a minimum of six hours. DNA was purified through ethanol precipitation and then used for ChIP-Seq. The DNA fragments were isolated from an agarose gel, blunt-ended, ligated to the Solexa adaptors, and sequenced using the Illumina 1G Genome Analyzer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Raw sequences generated from Illumina/Solexa sequencer were aligned using ELAND software to NCBI Build 36.1 (UCSC hg18) of the human genome. Only sequences that mapped uniquely to the genome with zero or one mismatch were used for further analysis. When multiple sequences mapped to the same genomic position, a maximum of two reads mapping to the same position were used. The sequenced reads represent only the ends of each immunoprecipitated fragments instead of the precise protein-DNA binding sites. To illustrate the entire DNA fragment, the 3’ end of each read was extended 200 bp. The reference genome was partitioned into 25 bp bins and the total reads (including partial reads) in each bin were summed and used to generate the visualization file in wiggle (WIG) format. The complete set of RefSeq genes was downloaded from the UCSC table browser on December 1, 2010. A custom annotated RefSeq gene list was generated by merging the all TSSs for each gene that were within 500 bases of each other. Then TSSs within 1000 bp of each other (6% of the total number) were removed from the list. This list was used for further analyses. A genomic coordinate file was compiled by extending the 3’ end of each original sequence to a total of 200 bp. The number of reads within 10,000 bases of the TSS of each gene in the custom gene list was tabulated. Similar analysis was applied to the location of center of peaks generated from the peak finding algorithm for the region within 500 bp relative to the TSS. Heat maps were generated using the program R. Genes were rank ordered based on the sequence density for RNAP II from -2K to +2K from the TSS. Using this order, basepair resolution sequence density for RNAP II and Gdown1 for the top 20,000 genes was displayed without binning. A peak finding algorithm (ChIP-Seq Peak) was designed to examine the data within individual WIG files and determine precise position and height of each significant peak. This algorithm assumes that each 200 bp immunoprecipitated DNA fragment is sequenced randomly from either end and that typical peaks would be approximately 400 bases wide at the base. Peaks less than 250 bp wide were eliminated and the center of each remaining peak was determined using Gaussian curve fitting. The height was determined by the area under each peak.
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| Submission date |
Feb 16, 2012 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Tiandao Li |
| Organization name |
Washington University
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| Street address |
4444 Forest Park Ave
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
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| Platform ID |
GPL9052 |
| Series (2) |
| GSE35878 |
Renormalization of GSE32442 and GSE33128 |
| GSE35886 |
Renormalization of GSE32442 and GSE33128 plus 1 control sample |
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| Relations |
| Reanalysis of |
GSM802743 |
| BioSample |
SAMN02197655 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM876981_PolII_60min_Flavo_HeLa_eland_norm.wig.gz |
75.9 Mb |
(ftp)(http) |
WIG |
| Processed data provided as supplementary file |
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