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Sample GSM538010 Query DataSets for GSM538010
Status Public on May 27, 2010
Title PUER-CEBPb-48h-ChIP-Seq
Sample type SRA
 
Source name PUER cells
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)
Treatment protocol Tamoxifen, 48h
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.
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
 
Description Chromatin IP against C/EBPb in PUER cells after 48h Tamoxifen treatment
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.
 
Submission date Apr 27, 2010
Last update date Jan 08, 2015
Contact name Christopher Benner
E-mail(s) cbenner@ucsd.edu
Organization name University of California, San Diego (UCSD)
Department Medicine
Street address 9500 Gilman Dr. MC 0640
City La Jolla
State/province California
ZIP/Postal code 92093-0640
Country USA
 
Platform ID GPL9185
Series (1)
GSE21512 Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities
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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