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Status |
Public on May 27, 2010 |
Title |
PUER-CEBPb-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: C/EBPb (sc-150) treatment: Tamoxifen, 48h transgenes: PU.1-ER strain: C57BL/6 genotype/variation: PU.1-/- chip antibody: C/EBPb (Vendor: Santa Cruz, cat# sc-150, lot# D0808)
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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 C/EBPb 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 |
SAMN02196095 |
Named Annotation |
GSM538010_Sample28.PUER-CEBPb-48h.bed.gz |
Supplementary file |
Size |
Download |
File type/resource |
GSM538010_Sample28.PUER-CEBPb-48h.bed.gz |
591.8 Kb |
(ftp)(http) |
BED |
GSM538010_Sample28.PUER-CEBPb-48h.fa.gz |
126.7 Mb |
(ftp)(http) |
FA |
Processed data provided as supplementary file |
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