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

large peritoneal macrophages

strain

C57BL6/J

treatment

4ng/ml Mcsf 7d, 2um DMSO 7d

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Microglia, LPMs, SPMs: After cell surface staining with antibodies required for cell sorting, pre-sorted cells samples containing microglia, or LPMs, and SPMs were fixed at room temperature with 1% paraformaldehyde/PBS containing 1mM butyric acid (Sigma) for 10 minutes. Reaction was then quenched by adding glycine to 0.125 M. Cells were then washed twice and sorted on BD Influx cell sorter, pelleted and snap-frozen in liquid nitrogen and stored at -80°C until ready for ChIP-Seq protocol preparation. RNA-Seq samples were immediately sorted after staining and not cross-linked. Cultured LPMs, TGEMs and BMDMs: Culture media was removed and cells were washed once in PBS at room temperature containing 1mM butyric acid (Sigma). Cells were then fixed at room temperature with 1% paraformaldehyde/PBS with 1mM butyric acid for 10 minutes. Reaction was then quenched by adding glycine to 0.125 M, and washed twice in PBS at room temperature. Cells were then scraped into the supernatant using a rubber policeman, pelleted, and snap-frozen in liquid nitrogen. RNA-Seq samples were not cross-linked. ChIPs for PU.1, H3K4me2, and H3K27Ac were performed as follow. 0.2e6-1.0e6 cells were incubated for 10 min in 1 ml (L1) 1.0% 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 Bioruptor Standard Sonicator in 200ml 10mM Tris/HCl pH 7.5, 100mM NaCl/0.5mM EGTA/0.1% Deoxcycholate/0.5% Sarkosyl for two cycles of 15min of 30sec on/60sec off each, on high settings. Triton-X to final concentration of 1% was then added, and lysates were cleared by centrifugation for 5min at 18,000 x g at 4°C. Supernatant was then immunoprecipitated for two hours at 4°C with antibody pre-bound to 20ml Protein A Dynabeads (Life Technologies) (PU.1: 3mg/sample, H3k4me2: 2ul from stock/sample, H3k27Ac: 1ul from stock/sample) Immunoprecipitates were washed three times each on ice with ice-cold wash buffer I (150 mM NaCl/1% Triton X-100/0.1% SDS/2mM EDTA), wash buffer III (10mM Tris/HCl/250 mM LiCl/1% IGEPAL CA-630/0.7% Deoxycholate/1mM EDTA) and TET (10mM Tris/HCl pH7.5/1mM EDTA/0.1% Tween-20), and eluted with 1% SDS/TE at room temperature in a final volume of 100ul. After addition of NaCl to 300 mM final Na+ concentration, crosslinks were reversed overnight at 65°C in a hot air oven, RNA digested for 1 h at 37°C with 0.33 mg/ml RNase A, proteins digested for 1 h at 55°C with 0.5 mg/ml proteinase K, and DNA extracted using Sera-Mag SpeedBeads (Thermo Scientific, cat no 6515205050250). To control for open chromatin and library biases, 1% input chromatin libraries after sonication were sequenced for each ChIP samples. These were also sampled before incubation with antibodies, reversed cross-linked and DNA collected as above. RNA-Seq: RNA was isolated by Trizol, precipitated with ethanol, treated with TurboDNASSE (Ambion/Life technologies cat. no.1907), precipitated with ethanol, and treated to either PolyA-selected RNA (MicroPoly(A)Purist kit (Ambion/Life Technologies cat. no. 1919)) or RiboZero rRNA removel kit (Epicentre cat. no. MRZH11124). ChIP-Seq: Sequencing libraries were prepared from collected DNA 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 12-15 cycles, size selected by gel extraction and sequenced on a Hi-Seq 2000 (Illumina) for 51 cycles. RNA-Seq: Enriched mRNA was hydrolyzed with Fragmentation Buffer (Ambion) for 10 min at 70°C and re-buffered to 10 mM Tris pH 7.4 using P-30 size exclusion columns (Bio-Rad). RNA was then de-capped for 2 h at 37°C with 0.5 µl (10 U/µl) TAP (Epicentre) in 20 µl TAP buffer containing 1 U/µl SUPERase-IN. Samples were 3’ dephosphorylated for 50’ at 37°C with 1 µl PNK (Enzymatics), 0.5 µl 10x TAP buffer, 1.5 µl water, 0.5 μl 0.25 M MgCl2 (3 mM free Mg2+ final), 0.5 μl 10 mM ATP (0.2 μM final to protect PNK). Subsequently, RNA was 5’-phosphorylated for 60 min at 37°C by adding 2 μl (10 U/µl) PNK, 10 μl 10x T4 DNA ligase buffer and 63 μl water. RNA was extracted with Trizol LS, precipitated in the presence of Glycoblue (Ambion), and dissolved in 4.5 µl water. 0.5 µl 9 µM of a 5’-adenylated sRNA3'MPX adapter /5Phos/AG ATC GGA AGA GCA CAC GTC TGA /3AmMO/ (IDT, desalted; adenylated with Mth ligase (NEB) according to the manufacturer’s instructions, phenol-chloroform/chloroform-extracted, ethanol-precipitated with glycogen and dissolved in water at 9 µM) were heat-denatured together with the RNA for 2 minutes at 70°C, and ligated with 100 U truncated T4RNA ligase 2 K227Q (NEB) in 10 µl 1x T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 hours at 16°C. To reduce adapter dimer formation, 0.5 μl 10 μM MPX_ RT primer 5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3’ (IDT, desalted) was added and annealed to the ligation product by incubating at 75°C for 2 minutes, then 37°C for 30 minutes, then 25°C for 15 minutes. Finally, 0.5 µl 5 µM hybrid DNA/RNA sRNA5'h adapter 5’-GTT CAG AGT TCT ACA rGrUrC rCrGrA rCrGrA rUrC-3’ (IDT) were ligated to previously capped RNA 5’ ends by adding 2 µl T4 RNA ligase buffer, 6 µl 50% PEG8000 (15% final), 1 µl 10 mM ATP, 9.5 µl water and 0.5 µl (5 U) T4 RNA ligase 1 for 90 minutes at 20°C. To 15 μl ligation reaction, an additional 0.5 μl 10 μM MPX_ RT primer were added, reactions were denatured at to 70°C for 1 minute, then placed on ice. RNA was reverse-transcribed by adding 3 µl 10x first strand buffer, 4.5 µl water, 1.5 µl 10 mM dNTP, 3 µl 0.1 M DTT, 1.5 µl RNaseOUT and 1 µl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. Complementary DNA was isolated by adding 35 µl AMPure XL beads (Beckman), binding and washing according to manufacturer’s instructions and dissolved in 40 µl TET. Libraries were PCR-amplified for 13 cycles with 0.75 µM primers oNTI201 (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAC G-3' and TruSeq-compatible indexed primers (e.g. 5’-CAA GCA GAA GAC GGC ATA CGA GAT iii iii GTG ACT GGA GTT CAG ACG TGT GCT CTT-3’ (desalted, IDT, i signifies index nucleotides) using Phusion Hot Start II (Thermo Scientific) in HF buffer containing 0.5 M betaine (98°C, 30s/12x(98°C, 10s/57°C, 25s/72°C, 20s)/ 72°C, 1min/4°C, ∞), 175-225 bp fragments were size-selected on 10% PAGE gels and sequenced for 51 cycles on a HiSeq 2000 sequencer (Illumina) with small RNA sequencing primer 5’-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3’ and TruSeq Index sequencing primer (Illumina).

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

79186080

Reads aligned (%)

98.0

Duplicates removed (%)

31.4

Number of peaks

589 (qval < 1E-05)

mm9

Number of total reads

79186080

Reads aligned (%)

97.9

Duplicates removed (%)

31.4

Number of peaks

578 (qval < 1E-05)