Antigen

Antigen Class

TFs and others

Antigen

Nr1h2

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

Sorted hepatic macrophages

strain

C57BL/6J

cell type

Sorted hepatic macrophages

cell subtype

F4/80-Hi Tim4-Pos Kupffer cells

treatment

NASH diet

chip antibody

LXR (Santa Cruz Biotechnology sc-1000X, RRID:AB_632067; sc-133221X, RRID:AB_2154783; sc-271064X, RRID:AB_10611071)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Hepatic macrophages (H3K27ac): Mice were humanely euthanized by exposure to CO2 and liver non-parenchymal cells processed for fluorescence activated cell sorting of Kupffer cells, with modifications from published methodology (Mederacke et al., 2015; Muse et al., 2018; Seki et al., 2007). In brief, livers were retrograde perfused for 3 min at a rate of 5-7 ml/min through the inferior vena cava with HBSS without Ca++ or Mg++ supplemented with 0.5 mM EGTA, 0.5 mM EDTA, and 20 mM HEPES. Perfusions were then switched to 40 ml of a digestion buffer, held at 37C, comprised of HBSS with Ca++ and Mg++ supplemented with 0.033 mg/ml of Liberase TM (Roche), 20 μg/ml DNaseI (Worthington), and 20 mM HEPES. Livers were then excised, minced, and digested for an additional 20 minutes in vitro at 37C with gentle rotation in 20 ml of fresh digestion buffer. The perfusion and digestion steps were performed in the presence of 1 μM flavopiridol to offset transcriptional changes associated with digestion. After tissue digestion, cells were passed through a 70-micron cell strainer and hepatocytes removed by 2 low-speed centrifugation steps at 50 X G for 2 min. Nonparenchymal cells in the supernatant were further separated from debris by pelleting for 15 min at 600 X G in 50 ml of 20% isotonic Percoll (Sigma Aldrich) at room temperature. Cells were then washed from Percoll containing buffer and suspended in 10 ml 28% OptiPrep (Sigma Aldrich) and carefully underlaid beneath 3 ml of wash buffer. The resulting gradient was centrifuged at 1,400 X G for 25 minutes at 4C with no break and cells enriched at the interface were saved and subjected to isotonic erythrocyte lysis. Enriched non-parenchymal cells were then washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Cells were then immunolabeled with specific antibodies of interest, washed, and sorted using a Beckman Coulter MoFlo Astrios EQ configured with spatially separated 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers. Kupffer cell nuclei (LXR ChIP): Mice were humanely euthanized by exposure to CO2, then briefly perfused with HBSS without Ca++ or Mg++ supplemented with 0.5mM EGTA, 0.5mM EDTA, and 20mM HEPES. Perfusions were then switched sequentially to 1 mg/ml disuccinimidyl glutarate in PBS for 30 minutes, then 1% formaldehyde in PBS for 10 minutes. Finally, the fixation was quenched by perfusion with 20 ml 0.125M glycine. Livers were excised, finely minced with a razor, and washed twice with 20 ml ice cold NF1 buffer (10mM Tris pH 8.0, 5mM MgCl2, 0.1M sucrose, 0.5% Triton X-100) followed by centrifugation 1,200 XG for 7 minutes at 4C. Liver pellets were next suspended in NF1 buffer and homogenized using the “loose” pestle of a Dounce homogenizer for 10 strokes, followed by incubation on ice for 30 minutes. Samples were finally homogenized using the “tight” pestle of a Dounce homogenizer for an additional 50-70 strokes, with periodic assessment for released nuclei by microscopy. The homogenized liver was then strained through a 70-micron mesh strainer, centrifuged at 1,200 XG for 7 minutes at 4C, then washed once more in PBS. Finally, the homogenized liver was suspended in PBS with 2mM EDTA and passed through a 40-micron mesh strainer. Nuclei were purified by FACS using a Sony SH800 based on TdTomato expression and forward scatter. Acquired nuclei were snap frozen in a dry ice ethanol bath and stored at -80C prior to use in ChIP-seq experiments. Peritoneal macrophages: As described in Gosselin et al. 2014, Cell PMID 25480297. ChIP protocol - H3K27ac: ChIP for H3K27ac was performed essentially as describe previously (Eichenfield et al., 2016). In brief, FACS purified cells were fixed with 1% paraformaldehyde for 10 minutes at room temperature. Next, 2.625 M glycine was added to 125 mM to quench fixation and cells were collected by centrifugation with the addition of 0.01% Tween-20 at 1,200 X G for 10 minutes at 4C. Cells were washed once with 0.01 % Tween-20 in PBS and collected by centrifugation at 1,200 X G for 10 minutes at 4C. Cell pellets were then snap frozen and stored at -80C. For ChIP reactions, cell pellets were thawed on ice and lysed in 80 μl LB3 (10 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5mM EGTA, 0.1% deoxcycholate, 0.5% sarkosyl, 1 × protease inhibitor cocktail, and 1 mM sodium butyrate). Lysate was sonicated using a Covaris for 12 cycles with the following setting: time, 60 seconds; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were collected and 10% Triton X-100 was added to 1% final concentration. One percent of the sonicated lysate was saved as a ChIP input. For each chromatin immunoprecipitation, aliquots of ~500,000 cells were added to 20 μl Dynabeads Protein A with 2 μg anti-H3K27ac (Active Motif) and incubated with slow rotation at 4C overnight. The following day, beads were collected using a magnet and washed three times each with wash buffer I (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, and 1 × protease inhibitor cocktail) and wash buffer III (10 mM Tris/HCl pH 7.5, 250 mM LiCl, 1% Triton X-100, 0.7% Deoxycholate, 1 mM EDTA, and 1 × protease inhibitor cocktail). Beads were then washed twice with ice cold 10 mM Tris/HCl pH 7.5, 1 mM EDTA, 0.2% Tween-20. Sequencing libraries were prepared for ChIP products while bound to the Dynabeads Protein A initially suspended in 25 μl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. ChIP protocol - LXR: For LXR ChIP-seq, FACS purified Kupffer cell nuclei were resuspended in wash buffer (10mM HEPES/KOH pH7.9, 85mM KCl, 1mM EDTA, 0.2% IGEPAL CA-630, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF) for 5 minutes on ice. Nuclei were spun down and resuspended in 130 μl RIPA-NR1 lysis buffer (20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF) and chromatin was sheared by sonication. Samples were sonicated in a 96 Place microTUBE Rack (Covaris cat#500282) using a Covaris E220 for 18 cycles with the following setting: time, 60 seconds; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were recovered and spun down at max speed, 4°C for 10 minutes. 1% supernatant was taken as input DNA and remaining supernatant was transferred to PCR strips and brought up to a volume of 200 μl using RIPA-NR1 lysis buffer (20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease 37 inhibitor cocktail (Sigma), 1 mM PMSF). 30 μl Dynabeads Protein A/G coated with 2 μg each of the indicated LXR specific antibodies (Santa Cruz Biotechnology: sc-1000X, sc- 133221X, sc-271064X) was added to the sample, and immunoprecipitation was carried out with slow rotation at 4°C overnight. Beads were then collected using a magnet and washed with 175 μl ice cold buffer as indicated by incubating samples on ice for 3 minutes: 3 times RIPA-NR1 lysis buffer (20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF), 6 times LiCl-NR1 buffer (10 mM Tris/HCl pH7.5, 250mM LiCl, 1 mM EDTA, 0.7% Na-Deoxycholate, 1% NP-40 alternative, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF), 3 times TET (10 mM Tris/HCl pH 8.0, 1 mM EDTA, 0.2% Tween-20, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF), and 1 time IDTE (10 mM Tris/HCl pH 8.0, 0.1 mM EDTA, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF). Bead complexes were resuspended in 25 μl TT (10 mM Tris/HCl pH 8.0, 0.05% Tween-20, 1x protease inhibitor cocktail) and sequencing libraries were prepared on-bead as described below. ChIP libraries were prepared while bound to Dynabeads using NEBNext Ultra II Library preparation kit with reaction

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

27308864

Reads aligned (%)

91.1

Duplicates removed (%)

31.3

Number of peaks

24186 (qval < 1E-05)

mm9

Number of total reads

27308864

Reads aligned (%)

91.0

Duplicates removed (%)

31.3

Number of peaks

24153 (qval < 1E-05)