Antigen

Antigen Class

Unclassified

Antigen

Unclassified

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

peritoneal thioglycolate-elicited macrophages

strain

BALBc/J

treatment

no treatment

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

ChIP-Seq: Media was decanted and cells were fixed at room temperature with either 1% formaldehyde/PBS for 10 minutes (for PU.1, C/EBPα ChIPs) or 2 mM disuccinimidylglutarate (DSG, Pierce)/10% DMSO/PBS for 30 minutes followed by 1% formaldehyde/PBS for another 15 minutes. After quenching the reaction by adding glycine to 0.125 M, cells were scraped into the supernatant using a rubber policeman, pelleted, washed twice with PBS and snap-frozen in dry-ice/methanol. ChIPs for PU.1 and C/EBPα were performed exactly as described previously (Heinz, S. et al. Molecular Cell 38, 576-589 (2010)). Briefly, 7e6-20e6 cells were incubated for 5 min in 1 ml 0.5% IGEPAL CA-630-containing 10 mM HEPES pH 7.9/85 mM KCl/1 mM EDTA on ice, centrifuged, sonicated on wet ice with a Misonix 3000 sonicator in 500 µl 1% SDS/0.5% EmpigenBB/50 mM Tris pH7.4/10 mM EDTA/1x protease inhibitor cocktail/1 mM PMSF for 6 cycles of 10s/30s break, lysates were cleared for 5 min, 20000 x g, 4°C, and the supernatant diluted with 1.5 volumes dilution buffer (0.5 % Triton X-100/20 mM Tris pH 7.4/100 mM NaCl/2 mM EDTA). Diluted chromatin was precleared for 1 h with 120 µl 50% slurry of CL-4B sepharose blocked (washed twice with TE buffer, blocked for 60 min in 0.5% BSA/20 µg/ml glycogen/TE, washed twice with TE, resuspended in original volume of TE), then immunoprecipitated overnight by adding 2.5 µg antibody, and capture with 50 µl protein A-sepharose (GE Healthcare Life Sciences, blocked as above, but overnight). Immunoprecipitates were washed twice each with ice-cold wash buffer I (150 mM NaCl/1% Triton X-100/0.1% SDS), II (450 mM NaCl/1% Triton X-100) , III (250 LiCl/1% IGEPAL CA-630/1% Deoxycholate) and TE, and eluted with 1% SDS/TE at room temperature. After addition of NaCl to 300 mM final Na+ concentration, crosslinks were reversed overnight in a hot air oven, RNA digested for 2 h at 37°C with 0.33 mg/ml RNase A, proteins digested for 2 h at 50°C with 0.5 mg/ml proteinase K, and DNA extracted using Qiagen QiaQuick columns. The H3K27Ac ChIPs were performed in the presence of 1mM (C57BL/6J and BALB/cJ) or 5 mM (CB6F1J) butyric acid in all buffers, using sepharose as described above for the C57BL/6J and BALB/c experiments, while for the CB6F1J experiment 5 µg antibody pre-bound to 50 µl Protein A Dynabeads (Life Technologies) were used, and the preclearing step was omitted. For p65, IP conditions were identical to the ones described before, except that pre-clearing was omitted, and the ChIP was performed with 5 µg antibody pre-bound to 50 µl Protein A Dynabeads for 30 minutes in 0.5% BSA, beads were washed thrice with WB I and thrice with WBIII containg only 0.7% deoxycholate, and a final wash with TE/50 mM NaCl was added before elution. ChIPs for H3K4me2 were performed on MNase-digested chromatin as described previously (Barski et al. Cell 129, 823-837 (2007)) on 20e6 cells digested in two 10e6 cell batches with 5 and 10 U MNase (Worthington) for 5 min at 37°C, s adding stop buffer containing 10 mM each of EDTA/EGTA, brief sonication in a Bioruptor 200, dialysis against two changes of RIPA buffer, and overnight IP with antibody-bound, block protein A-Dynabeads (Life Technologies) at 4°C. After two washes each with RIPA buffer, 0.3 M NaCl/RIPA buffer, 0.5% deoxycholate/0.5% IGEPAL CA-630-containing buffer and TE, DNA was eluted with 0.3% SDS/TE and 0.9 mg/ml proteinase K overnight at 65°C overnight, and DNA was isolated by phenol/chloroform extraction and ethanol precipitation. To control for open chromatin and library biases, input chromatin libraries after sonication were sequenced for each strain, crosslinking condition and ChIP lysis protocol. Sequencing libraries were prepared by blunting, A-tailing, adapter ligation as previously described (Heinz, S. et al. Molecular Cell 38, 576-589 (2010)) using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 12-14 cycles, size selected by gel extraction and sequenced on a Hi-Seq 2000 (Illumina) for 51 cycles.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

10285254

Reads aligned (%)

94.4

Duplicates removed (%)

28.3

Number of peaks

30489 (qval < 1E-05)

mm9

Number of total reads

10285254

Reads aligned (%)

94.2

Duplicates removed (%)

28.4

Number of peaks

30521 (qval < 1E-05)