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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 27, 2010 |
| Title |
PUER-PU.1-48h-ChIP-Seq |
| Sample type |
SRA |
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| Source name |
PUER cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: PUER cells
chip antibody: PU.1 (sc-352)
treatment: Tamoxifen, 48h
transgenes: PU.1-ER
strain: C57BL/6
genotype/variation: PU.1-/-
chip antibody: PU.1 (Vendor: Santa Cruz, cat# sc-352, lot# F1808)
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| Treatment protocol |
Tamoxifen, 48h
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| Growth protocol |
PUER cells were propagated in 10 % FCS/Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 100 U/ml penicillin/streptomycin, 1 µg/ml puromycin, 2 mM L-glutamine and 5 ng/ml recombinant mIL-4. The PU.1-ER fusion protein was activated by adding 4-hydroxy-tamoxifen at a final concentration of 100 nM to the media.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody. Alternatively, DNA was phenol/chloroform-extracted from MNase-treated nuclei, and 134-154 bp long fragments were extractred from a 2% agarose gel. 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, ChIP DNA was PCR-amplified with Illumina primers for 12-16 cycles and library fragments of 200-300 bp (ChIP) (insert plus adaptor and PCR primer sequences) were isolated from a 2% agarose gel. For MNase-Seq, size-selected mononucleosome-derived DNA fragments were ligated to modified adapters that incorporate both the adapter sequence as well as the primer landing sites for the flow cell-conjugated primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer or Genome Analyzer II following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Chromatin IP against PU.1 in PUER cells after 48h Tamoxifen treatment
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| Data processing |
Alignment: Reads were truncated to 25 bp and aligned to the mouse mm8 genome (NCBI Build 36) using eland. Only reads that mapped to a single, unique position were used for downstream analysis.
Peak Identification: Peaks were identified using custom software (HOMER, available at http://biowhat.ucsd.edu/homer/). For transcription factors, peaks were found by identifying 200 bp regions with tag density exceeding a threshold set at a 0.1% false discovery rate (determined by tag randomization). Only peaks with >4x normalized tag density relative to control sequencing and >4x normlaized tag density to the sourrounding 10kb of region were considered for analysis.
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| Submission date |
Apr 27, 2010 |
| Last update date |
Jan 08, 2015 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
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| Organization name |
University of California, San Diego (UCSD)
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| Department |
Medicine
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| Street address |
9500 Gilman Dr. MC 0640
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE21512 |
Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities |
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| Relations |
| BioSample |
SAMN02196120 |
| Named Annotation |
GSM538004_Sample22.PUER-PU.1-48h.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM538004_Sample22.PUER-PU.1-48h.bed.gz |
673.0 Kb |
(ftp)(http) |
BED |
| GSM538004_Sample22.PUER-PU.1-48h.fa.gz |
109.2 Mb |
(ftp)(http) |
FA |
| Processed data provided as supplementary file |
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