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| Status |
Public on Feb 01, 2014 |
| Title |
Pol II ChIP-Seq in HeLa |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
antibody: Pol II (N-20) (Santa Cruz, sc-899)
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| Growth protocol |
HeLa cells were maintained in suspension at 5% CO2 and 37°C in Minimum Essential Medium Eagle Spinner Modification (S-MEM) (Gibco, 11380-037) supplemented with 2 mM L-glutamine and 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HeLa cells were grown to 0.5 million cells/ml and crosslinked with 1% formaldehyde in media for 10 minutes before glycine addition to 125 mM. After washing and lysis, cells were sonicated in RIPA buffer (50 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 0.25% sodium deoxycholate; 1% IGEPAL CA-630) on ice using a Fisher Model 550 Sonic Dismembrator. For each ChIP, sonicated supernatant from 5 million cells was incubated at 4°C overnight with 10 µg of Pol II (Santa Cruz, sc-899), NELF (sc-23599), or DSIF (sc-28678) antibody, and then 1 hr with 50 µl Protein G-Sepharose bead slurry (Sigma, P3296). Beads were washed in columns with 15 ml ice cold RIPA buffer and rinsed with 10 ml ice cold PBS. Immunocomplexes were eluted, incubated at 65°C overnight to reverse crosslinks, and treated with RNase A and Proteinase K. DNA was isolated by MinElute PCR purification kit (QIAGEN, 28004).
Library preparation was performed by the University of Iowa DNA Facility using the Ovation SP Ultralow DR Multiplex System (Nugen, 8033-32) for Mondrian.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
base-calling was performed by the University of Iowa DNA Facility
paired-end ChIP-Seq reads were filtered for fragments between 50-500 bp and aligned to hg19 using Bowtie 2.0.5 and the following parameters: --minins 50 --maxins 500 --no-mixed --no-discordant
alignment output was converted from sam to bampe format using Samtools 0.1.18
bedGraph files for visualizing ChIP-Seq reads were generated using MACS 2.0.10 "callpeak" function and the following parameters: --format BAMPE --bdg
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files were generated as described above using MACS 2.0.10; scores represent the sum of overlapping ChIP fragments over hg19
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| Submission date |
Dec 05, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
David Price |
| E-mail(s) |
david-price@uiowa.edu
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| Organization name |
University of Iowa
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| Department |
Biochemistry
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| Lab |
260 EMRB
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| Street address |
500 Newton Road
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE53008 |
Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells |
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| Relations |
| BioSample |
SAMN02437198 |
| SRA |
SRX387510 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1280297_PolII-50-500.bedgraph.gz |
166.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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