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Sample GSM538017 Query DataSets for GSM538017
Status Public on May 27, 2010
Title BMDM-PU.1-ChIP-Seq
Sample type SRA
 
Source name bone marrow-derived macrophages
Organism Mus musculus
Characteristics cell type: macrophage
chip antibody: PU.1 (sc-352)
treatment: none
transgenes: none
strain: C57BL/6
genotype/variation: wild-type
chip antibody: PU.1 (Vendor: Santa Cruz, cat# sc-352, lot# F1808)
Treatment protocol none
Growth protocol Bone marrow was harvested and washed once with PBS, seeded/cultured in DMEM containing 20 % FCS, 30 % L-cell conditioned media (as source of macrophage colony stimulating factor, M-CSF), and 100 U/ml penicillin/streptomycin in non-tissue culture-treated petri dishes. After 6-8 days of culture, non-adherent cells were washed off with room temperature PBS and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in RPMI 1640 containing 10% FCS, 100 U/ml penicillin/streptomycin, and 10ng/ml M-CSF.
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 II
 
Description Chromatin IP against PU.1 in BMDM
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 May 15, 2019
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 GPL9250
Series (1)
GSE21512 Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities
Relations
SRA SRX019780
BioSample SAMN00012142
Named Annotation GSM538017_Sample37.BMDM-PU.1.bed.gz

Supplementary file Size Download File type/resource
GSM538017_Sample37.BMDM-PU.1.bed.gz 1.1 Mb (ftp)(http) BED
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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