Antigen

Antigen Class

TFs and others

Antigen

Rela

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

BMM_LPS_0120_P65

strain

C57BL/6

genotype/variation

wild type

age

8-12 weeks

tissue

primary murine bone marrow macrophage cells (BMMs)

stimuated with

lipopolysaccharide (LPS)

time after lps stimulation

120 minutes

chip antibody

p65

chip antibody vendor

Santa Cruz Biotechnology Inc.

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP–Seq analysis, formaldehyde-fixed cells were sonicated and processed for immunoprecipitation. In brief, 3 × 107 BMMs were fixed for 10 min in 1% formaldehyde in PBS, washed in PBS, and harvested by scraping in PBS. Cells were lysed by resuspending cell pellets in RIPA buffer (10mM Tris-HCl, pH8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% Deoxcholic acid sodium salt) and drawing the cell suspensions three times through a 30 gauge needle. Chromatin was sheared using a probe sonicator (130 W Ultrasonic Processor with a 3mm tip, 5 × 60s at 30% maximum setting). Sonication quality was checked by gel electrophoresis. Protein concentrations in the extracts were determined (BioRad DC Protein Assay Kit I # 500-111) and sonicated cell extracts containing 0.5 mg protein were incubated with antibodies overnight at 4oC. Immune complexes were recovered by incubation with a 50%-50% mix of magnetic beads coated with Protein A or Protein G (Dynabeads Protein A Invitrogen # 100.02D, Dynabeads Protein G Invitrogen # 100.04D). The magnetic beads were washed with RIPA buffer and the chromatin was eluted with 1% SDS in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0) at 65oC for 15 min. Eluted chromatin was reverse-cross-linked by adding 226 mM NaCl and incubating at 65oC for > 5 hr. Then 36 mM Tris–HCl pH8.0, 9 mM EDTA, and 1.5 U of Proteinase K (Fermentas # EO0491) was added to the samples and they were incubated at 42oC for > 1 hr. DNA was purified using phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) extraction. The purified immunoprecipitated DNA was prepared for sequencing with the Illumina ChIP-Seq Sample Prep Kit and processed according to the manufacturer’s instructions (Illumina Part # 11257047 Rev. A). A sequencing library for the Illumina Genome Analyzer was derived from the IP using the Illumina reagent kit (see systemsimmunology.org). Single-ended, 36-cycle sequencing was performed on an Illumina Genome Analyzer, and the raw image data were processed using the Illumina Genome Analysis Pipeline Software on a dedicated sequence data processing system (see Genome Analyzer Pipeline Software User Guide, Illumina, San Diego, CA, USA, v0.3).

Sequencing Platform

instrument_model

Illumina Genome Analyzer

mm10

Number of total reads

14561360

Reads aligned (%)

87.9

Duplicates removed (%)

17.9

Number of peaks

715 (qval < 1E-05)

mm9

Number of total reads

14561360

Reads aligned (%)

87.7

Duplicates removed (%)

18.1

Number of peaks

815 (qval < 1E-05)