|
| Status |
Public on Nov 25, 2009 |
| Title |
K562_GATA1_ChIP |
| Sample type |
SRA |
| |
|
| Source name |
Chromatin IP against GATA1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
chip antibody: GATA1
|
| Treatment protocol |
N/A
|
| Growth protocol |
Human K562 erythroleukemia cells (ATCC # CCL-243) were cultured in RPMI medium 1640 supplemented with 10% heat inactivated FBS and antibiotic-antimycotic solution (Invitrogen, 15240-104) in a 36-38°C, 5-10% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP samples were prepared using 10e8 K562 cells per condition. Chromatin was prepared as described (http://www.genomecenter.ucdavis.edu/farnham/protocol.html). ChIP libraries were created using 15 cycles of amplification. Libraries were run on a 2% agarose gel, and the 150-450 bp fraction was extracted and purified using DNA gel extraction kit (QIAGEN). To estimate the yield of library and its relative amplification value, library DNA was quantitated using a Nanodrop, and serial dilutions of 1.25 nM library were compared to a reference library by real-time PCR using primers complementary to the adapters. Real-time PCR was performed using SYBR® Green Master PCR Mix (Invitrogen). The amplification C(t) value relative to a known reference library was used to estimate the flowcell loading concentration. The ChIP-seq libraries were run on an Illumina GA2 at the U. California-Davis.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Rep1+Rep2
|
| Data processing |
Alignment: Sequence reads obtained were aligned to the UCSC human genome assembly HG18 using the Eland application (Illumina), allowing no more than two mismatches per sequence. Only sequences that aligned uniquely were used to identify GATA-1 and GATA-2 occupancy peaks. The 32 nt sequence reads were extended by the average length of the ChIP-seq library (327 bp).
Peaks: Binding sites were determined using a multi-pass system. The first pass of the program determines an accurate, statistically significant height cutoff for peaks. Sequenceable regions of the genome are estimated based on 12 input sample datasets of K562 and HeLa cells (unpublished data). This approach accounts for genomic regions with a lower abundance of sequence reads and provides a basis for randomizing sequence reads from the ChIP-seq experiments. The minimum peak height is then determined, using a false discovery rate of 0.001. The second pass eliminates false peaks that are also present in input data sets. The background model was produced by randomly selecting sequence reads from input libraries and placed in ten files of equal size. Both the ChIP-seq and input data sets are binned using a sliding window of 30 bp. ChIP sample bins that are above height cutoff and are significant vs. input background, using a one sample T-test, are assessed as potential peaks. A third pass to the peak detection removes peaks in duplicated regions. The resulting potential peaks with lengths greater than the average length of chromatin fragments used in the ChIP experiment are reported. The height of a peak reflects the number of overlapped sequence reads for that peak.
|
| |
|
| Submission date |
Nov 03, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Emery H. Bresnick |
| E-mail(s) |
[email protected]
|
| Phone |
608-265-6446
|
| Fax |
608-262-1257
|
| URL |
http://www.wisc.edu/molpharm/faculty/bresnick.html
|
| Organization name |
University of Wisconsin-Madison
|
| Department |
Pharmacology
|
| Lab |
Bresnick Lab
|
| Street address |
1300 University Avenue 383 MSC
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE18829 |
Discovering Hematopoietic Mechanisms Through Genome-Wide Analysis of GATA Factor Chromatin Occupancy |
| GSE18868 |
Genome-wide maps of GATA factor occupancy in K562 cells |
|
| Relations |
| Reanalyzed by |
GSE59801 |
| SRA |
SRX014810 |
| BioSample |
SAMN00006991 |
