Antigen

Antigen Class

TFs and others

Antigen

Mafb

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

bone marrow derived macrophages

strain

C57BL/6J

genotype

wild type

chip antibody

MafB (Bethyl IHC-101)

cell type

bone marrow derived macrophages

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cells cultured in plates were fixed by the addition of 1/10 volume of freshly made cross-linking solution (11% formaldehyde, 100mM NaCl, 1mM EDTA, 0.5 mM EGTA, 50mM Hepes pH 7.8) directly to cell medium and incubation at room temperature (RT) for 10 minutes. Formaldehyde was then quenched for 5 minutes at RT by the addition of 2.5M Glycine solution to a final concentration of 125mM. Fixed cells were washed twice with cold PBS, scraped off the plate, counted, and transferred in 50ml Falcon tubes. Cells were then pelleted by centrifugation at 700g for 5 minutes at 4oC, snap-frozen in liquid nitrogen and stored at -80oC for storage or shipment on dry-ice. Each batch of 100 millions cells was lysed by adding 10 ml of ChIP Lysis Buffer (Santa Cruz Biotechnology, sc-45000) or RIPA buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing Protease Inhibitor Cocktail (PIC). One complete PIC tablet (Roche, REF 11873580001) for 50 ml buffer or 1 mini PIC tablet (Roche, REF 11836153001) for 10ml buffer was used. After rotating the cell tube for 10-15 minutes at 4oC, nuclei were pelleted by centrifugation at 700g for 5 minutes at 4oC. Nuclei pellet was resuspended in 5ml of Santa Cruz Biotechnology ChIP Lysis Buffer High Salt (sc-45001) or RIPA buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing PIC. Aliquots of 1ml containing 20 million nuclei were transferred into 1.5 ml low-bind microfuge tubes. The nuclei suspension was either sonicated immediately or snap-frozen in liquid nitrogen and stored at -80oC. The nuclei/chromatin suspension in each ml was sonicated (Sonics Vibra Cell, Model CV188, with Stepped Tip 1/8”-630-0422) on ice in 4oC cold room. Sonication was performed with 9 cycles of 30 seconds (s) ON at 60% intensity and 30 s OFF for chromosome modifications, H3k4me1 and H3K27ac, or with 8 cycles for transcription factors, P300 and PU.1. The sheared chromatin was centrifuged at 14krpm for 10-15 minutes, then transferred as in the supernatant into a new 1.5 tube and kept on ice for ChIP. For ChIP, 50 ul Invitrogen Dynabeads (7x108 beads/ml) were used for 20 millions cells, either anti-rabbit IgG (Cat. no. 112.03D) or anti-mouse IgG (Cat. no. 112.01D) depending on the source of the first antibody. The beads were first washed with PBS/BSA/PIC buffer (1x PBS, 5mg/ml BSA (Sigma, A3059-10G, Fraction V), Roche PIC (1 mini tablet for 10 ml or 1 complete tablet for 50 ml, added before use)) then washed beads were thoroughly resuspended in 1ml of PBS/BSA/PIC buffer in a 1.5 ml microfuge tube. Tube was placed on a magnetic rack for 1 minute, then the supernatant was removed. Wash was repeated twice by resuspending beads in 1 ml PBS/BSA/PIC buffer, rotating for 5 minutes and supernatant was removed as above. The beads were then coated with antibody by resuspending in 250 ul PBS/BSA/PIC buffer in each tube, adding 5 ug antibody, rotating overnight at 4 oC. The following antibodies were used: H3K4me1 (Abcam, ab8895), H3K4m3 (Abcam ab8580), H3K27ac (Abcam, ab4729), P300 (Santa Cruz Biotechnology, sc-585), PU.1 (Santa Cruz Biotechnology, sc-352), MafB (Bethyl IHC-101) and Flag M2 antibody (Sigma). After overnight incubation with antibody, the beads were pelleted by placing the tube on a magnetic rack for 1 minute followed by removing the supernatant. The beads were then washed 3 times with PBS/BSA/PIC buffer. ChIP was carried out by adding the sheared chromatin suspension to the antibody-coated beads, rotating overnight at 4 oC, and followed by a series of washes. The beads were first washed once with 1ml of low salt wash Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), then twice with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), three times with LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% GEPAL CA630, 1% Na-Deoxyholate), and twice with TE buffer. ChIP DNA was eluted by adding 200 ul of ChIP Elution Buffer (Santa Cruz Biotechnology, sc-45003, or 1%SDS/0.1 M NaHCO3.) to each bead tube, vortexing to resuspend beads, and incubation at 65oC for 1 hour with vortexing every 15 minutes. After centrifugation for 3 minutes at RT and placing on a magnetic rack for 1 minute, ChIP DNA in the supernatant was transferred to a new tube. Reversing of crosslinks was done by incubation at 65oC overnight, and DNA was extracted with an equal volume of Phenol:Chloroform:IAA (24:24:1). After centrifugation at 14krpm for 3 minutes, ChIP DNA in the supernatant was transferred to a 2.0 ml low-bind tube. More DNA was extracted by adding 100 ul water to the Phenol:Chloroform phase and repeating the exaction. DNA extract was combined and mixed with 5 volumes of PBI (from Qiagen MinElute PCR Purification Kit, Cat# 28004). The pH of the mixture was adjusted with 3M NaAc pH5.2 to lower than 7.5 before apply to the column. The mixture turned back to yellow color, which is important for DNA to bind to Qiagen column. DNA was purified according to manufacturer's instructions, and eluted in 30 ul buffer EB. DNA concentration was measured with Qubit. 30ng (P300, PU.1, MafB and Flag-MafB) to 100ng (H3K4me1 and H3K27ac) DNA were used to make each library. ChIP-seq libraries were made using adaptors from Illumina (FC102-1001) and other enzymes and reagents from NEB, following the Illumina ChIP-seq protocol with some minor modifications. ChIP DNA was end repaired by T4 DNA polymerase (M0203S/L), Klenow DNA polymerase (M0210S/L), and T4 PNK (M0201S/L), and purified with Qiagen MinElute PCR Purification Kit. A-base was then added to the 3' end by Klenow Fragment, 3' to 5' exo- (NEB, Cat# M0212S/L) and dATP. The Illumina adaptors (1ul of 1:10 dilution) were subsequently added to both ends by DNA Ligase (M2200S/L). The adaptor-ligated DNA was sized selected for 200-400bp fragments by 2% low-melting agarose gel (Lonza, Cat# 50080), followed by purification by Qiagen MinElute Gel Extraction Kit (Cat# 28604). Library DNA was amplified in 100 ul reaction by Phusion PCR Master Mix (NEB, Cat# F-531S), primers SolexaPCR_F (5'- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T -3') and SolexaPCR_R (5'- CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T -3'). Libraries of H3K4me1 and H3K27ac were amplified by pre-denaturing at 98C for 30 sec followed by 10 cycles of (98C / 10s, 65C / 30s 72C / 30s), and extra 5 min at 72C at the end. For the P300 and PU.1 libraries, 12 cycles of PCR were used. After library DNA was purified, library concentration was measured by Qubit, and size distribution was determined by Bioanalyzer. Each ChIP-seq library was sequenced on 1 lane of Illumina GAIIx or Illumina HiSeq 2000 with 1x36 bases read length.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

26631284

Reads aligned (%)

95.1

Duplicates removed (%)

12.2

Number of peaks

24472 (qval < 1E-05)

mm9

Number of total reads

26631284

Reads aligned (%)

94.9

Duplicates removed (%)

12.3

Number of peaks

24466 (qval < 1E-05)