Antigen

Antigen Class

TFs and others

Antigen

Pparg

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

Primary thioglycollate-elicited peritoneal macrophages

strain

C57BL6

chip target

PPARg

chip antibody

sc-7196, sc-1984

genotype

wild-type

cell type

macrophages

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

20-40 million macrophages were used per immunoprecipitation. Cells were first crosslinked in 2mM disuccinimidyl glutarate (Thermo Fisher Scientific, Rockford, IL, USA) in PBS for 30 minutes, then subsequently in 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 minutes all at room temperature. The reactions were quenched by adding glycine (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 125 mM, and the cells were centrifuged and washed twice with ice-cold PBS and swollen for 10 min on ice in hypotonic buffer. Nuclei were then pelleted and resuspended in RIPA lysis buffer (10mM Tris/HCl, 1mM EDTA, 1mM EGTA, 0.1% SDS, 0.5% Na-Deoxycholate, 1% NP-40, 150mM NaCl). Chromatin was sheared to an average DNA size of 100-400 bp by administering 10 pulses of 30 seconds duration at 12 W power output with 1 min pause on wet ice using a Misonix 3000 sonicator. Extracts were clarified by centrifugation at 14,000 rpm for 15 min at 4ºC, pre-cleared with 100 μl of CL4B sepharose beads (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at 4ºC, then incubated overnight with appropriate specific antibody on rotator. Protein-DNA complexes bound to antibodies were collected by incubation with 40 μl of Protein A beads (GE Healthcare, Pittsburgh, PA, USA) for 2 hr at 4ºC on rotator. Beads were washed 3 times with RIPA lysis buffer, 6 times with LiCl buffer (100mM Tris/HCl, 500mM LiCl, 1% NP-40, 1% Na-Deoxycholate, 1mM EDTA) and twice with TE. Immunoprecipitated chromatin was eluted twice with 100 μl elution buffer (0.1M NaHCO3, 1% SDS) followed by overnight incubation at 65ºC adding NaCl. Samples were treated with RNase A and proteinase K. DNA was isolated using the ChIP DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instruction. Libraries were prepared according to Illumina's instructions. 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 (minus exo) 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 Genomic adaptors or with NEXTflex DNA barcodes adaptors for 18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size-selected (150-250bp) from a 2% agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Illumina Genome Analyzer II or Illumina HiSeq 2000 according to the manuactuere’s instructions.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

18484526

Reads aligned (%)

91.6

Duplicates removed (%)

39.4

Number of peaks

19408 (qval < 1E-05)

mm9

Number of total reads

18484526

Reads aligned (%)

91.5

Duplicates removed (%)

39.6

Number of peaks

19415 (qval < 1E-05)