Antigen

Antigen Class

TFs and others

Antigen

Nr4a1

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

pINDUCER20b-HA-NUR77 RAW264.7 macrophages

cell line

RAW264.7

transgene

pINDUCER20b-HA-NUR77

treatment

1 µg/mL Dox for 18 hours

number of cells

12 x 10^7

chip antibody

Mouse anti-HA tag (Diagenode, catalog# C15200190, lot# 001)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cells were washed with PBS and dual crosslinked directly in culture plates with 2 mM Di(N-succinimidyl) glutarate (Sigma) in PBS for 45 minutes at room temperature, followed by 1% formaldehyde (Sigma) for 15 minutes at room temperature. Crosslinking reactions were quenched by addition of glycine to 0.125 M final concentration and incubation for 5 minutes at room temperature. Crosslinked cells were washed with ice-cold PBS and collected by scraping in ice-cold PBS followed by centrifugation for 5 minutes at 500 x g. Cell pellets were resuspended in resuspension buffer L1A (10 mM HEPES-KOH pH 7.9, 85 mM KCl, 1 mM EDTA). Cell membranes were lysed by diluting samples 1:1 with lysis buffer L1B (L1A plus 1% IGEPAL CA-630) and incubating 10 minutes on ice. Cells were subsequently centrifuged 5 minutes at 700 x g and pellets were resuspended in lysis buffer L2 (50 mM Tris-HCl pH 7.4, 1% SDS, 10 mM EDTA). Nuclei were ruptured and chromatin was sonicated into approximately 200 bp fragments using a Bioruptor Pico (Diagenode) for 20 cycles of 30 seconds on and 30 seconds off. Fragment length was confirmed by running an aliquot of reverse crosslinked chromatin on a 1.2% agarose gel. Sonicated chromatin samples were centrifuged for 10 minutes at 16,000 x g to pellet cell debris. Supernatants were transferred to DNA LoBind tubes (Sigma) and diluted 1:1.5 with dilution buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100). Per ChIP reaction, 30 µL Protein G Dynabeads (Novex) were washed twice with 0.5% bovine serum albumin (BSA) in PBS and incubated with 2 µg anti-HA antibody (Diagenode) for 3 hours at 4°C. Dynabead-antibody complexes were added to each diluted chromatin sample (18 ChIPs total) and incubated overnight at 4°C to immunoprecipitate chromatin-linked HA-NUR77 protein. Immunoprecipitates were washed four times with wash buffer 1 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), four times with wash buffer 2 (10 mM Tris-HCl pH 7.4, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% sodium deoxycholate, 1 mM EDTA), twice with wash buffer 3 (10 mM Tris-HCl pH 7.4, 0.2% Triton X-100, 1 mM EDTA), and twice with wash buffer 4 (10 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA). Samples were transferred to fresh DNA LoBind tubes after the first and second to last wash steps to reduce background. Chromatin was eluted from Dynabeads by incubating in elution buffer (10 mM Tris-HCl pH 7.4, 2% SDS, 1 mM EDTA) for 20 minutes at 37°C. Crosslinks were reversed by addition of NaCl to a final concentration of 300 mM and incubating overnight at 65°C. DNA was purified by incubating samples with 0.33 mg/mL RNase A for 1 hour at 37°C, followed by 0.5 mg/mL Proteinase K for 1 hour at 50°C, and extraction of DNA by phenol:chloroform:isoamylalcohol precipitation. Precipitated DNA was dissolved in ultrapure water and DNA from 6 separate ChIP reactions was pooled to create one biological replicate, resulting in three biological replicates total. ChIP DNA libraries were prepared as previously described (Schmidt et al., 2009; PMID: 19275939).

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

30324115

Reads aligned (%)

44.9

Duplicates removed (%)

16.1

Number of peaks

2136 (qval < 1E-05)

mm9

Number of total reads

30324115

Reads aligned (%)

44.8

Duplicates removed (%)

16.2

Number of peaks

2132 (qval < 1E-05)