Antigen

Antigen Class

TFs and others

Antigen

Spi1

Cell type

Cell type Class

Blood

Cell type

Macrophages

MeSH Description

The relatively long-lived phagocytic cell of mammalian tissues that are derived from blood MONOCYTES. Main types are PERITONEAL MACROPHAGES; ALVEOLAR MACROPHAGES; HISTIOCYTES; KUPFFER CELLS of the liver; and OSTEOCLASTS. They may further differentiate within chronic inflammatory lesions to EPITHELIOID CELLS or may fuse to form FOREIGN BODY GIANT CELLS or LANGHANS GIANT CELLS. (from The Dictionary of Cell Biology, Lackie and Dow, 3rd ed.)

Sample

source_name

In vitro reconstituted chromatin (genomic DNA from primary bone marrow-derived macrophages)

strain

C57BL/6

cell type

BMDM cells (7th day of differentiation)

antibody

Anti-PU.1 (in-house rabbit polyclonal against the N-Terminal (aa. 1-100))

retroviral infection

No treatment

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Naked genomic DNA was purified from bone marrow derived murine macrophages by three consecutive phenol/chloroform extractions. DNA was sonicated to obtain fragments smaller than 2 kb, and fragments ranging from 600 to 2000 kb were purified with Solid-Phase Reversible Immobilization (SPRI) beads (Agencourt AMPure XP, Beckman Coulter). DNA was combined with recombinant histones (EpiMark Nucleosome Assembly Kit, NEB E5350) to generate nucleosomes by salt dialysis (PMID: 10372352). DNA molecules were considered as multiple of 150 bp nucleosome-assembling units. Assembly reaction was performed mixing octamers and nucleosome-assembling units in a molar ratio 1:2, such that DNA was not limiting and octamer would assemble according to the sequence preference. In vitro nucleosomes were partially digested with MNase (5U for 2 minutes in the digestion buffer described above) to obtain mainly di- and tri-nucleosomes and to eliminate any residual unwrapped DNA. They were then incubated with macrophage-derived nuclear extracts. Nuclear extracts were prepared from 2e7 cells. Cells were first lysed with hypotonic buffer (10 mM Tris-HCl, 1 mM KCl, 1.5 mM MgCl2), then nuclei were lysed with a high-salt buffer (50 mM Tris-HCl, 200 mM NaCl, 10% glycerol, 0.2% NP40) and diluted 1:2 with a dilution buffer (10 mM Tris-HCl, 2 mM EDTA). Nuclear extracts were subjected twice (2 hours and overnight) to immunodepletion with 8 μg of Pu.1 antibody or normal rabbit IgG. Incubation of nuclear extracts and in vitro nucleosomes was performed at 4°C for 2 hours, then 5 μg of anti-Pu.1 antibody were added for 1 hour and DNA-protein complexes recovered with G protein-coupled magnetic beads. Beads were washed 6 times with wash buffer (30 mM Tris-HCl, 200 mM NaCl, 10% glycerol, 0.1% NP40, 1 mM EDTA) and twice with TE. DNA was eluted in TE-2% SDS. DNA was then purified by Qiaquick PCR purification kit and quantified with PicoGreen (Invitrogen). ChIP DNA was prepared for HiSeq2000 sequencing following standard protocols.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

38740809

Reads aligned (%)

90.4

Duplicates removed (%)

27.5

Number of peaks

589 (qval < 1E-05)

mm9

Number of total reads

38740809

Reads aligned (%)

90.0

Duplicates removed (%)

27.4

Number of peaks

630 (qval < 1E-05)