Antigen

Antigen Class

ATAC-Seq

Antigen

ATAC-Seq

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 macropages (BMDM)

strain

NOD/ShiLtJ

tissue

bone marrow derived macropages (BMDM)

culture protocol

7 day cultured BMDMs

ligands in culture

KLA 1h

chip antibody

NA

Sequenced DNA Library

library_strategy

ATAC-seq

library_source

GENOMIC

library_selection

other

library_construction_protocol

Femur, tibia and iliac bones from the different mouse strains were flushed with DMEM high glucose (Corning) and red blood cells were lysed using red blood cell lysis buffer (eBioscience). After counting, 20 million bone marrow cells were seeded per 15cm non-tissue culture plates in DMEM high glucose (50%) with 20% FBS (Omega Biosciences), 30% L929-cell conditioned media (as source of M-CSF), 100 U/ml penicillin/streptomycin+L-glutamine (Gibco) and 2.5μg/ml Amphotericin B (HyClone). After 4 days of differentiation, 16.7 ng/ml mouse M-CSF (Shenandoah Biotechnology) was added to the media. After an additional 2 days of culture, non-adherent cells were washed off with room temperature DMEM high glucose and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in DMEM high glucose containing 10% FBS, 100 U/ml penicillin/streptomycin+L-glutamine, 2.5μg/ml Amphotericin B and 16.7 ng/ml M-CSF. For KLA activation, macrophages were treated with 10 ng/mL KLA (Avanti Polar Lipids) for 1 or 6 hours. ATAC-seq: To approximately 150k nuclei in 22.5ul GRO freezing buffer, isolated as described for GRO-seq above, 25ul DNA Tagmentation buffer was added, reaction mixed and 2.5uL DNA Tagmentation Enzyme mix (Nextera DNA Library Preparation Kit, Illumina) added. Mixture was incubated at 37°C for 30 minutes and subsequently purified using the Zymogen ChIP DNA purification kit as described by the manufacturer. DNA was amplified using the Nextera Primer Ad1 and a unique Ad2.n barcoding primers using NEBNext High-Fidelity 2X PCR MM for 10 cycles. PCR reactions were purified using 1.5 volumes of SpeedBeads in 2.5M NaCl, 20% PEG8000, size selected using PAGE/TBE gels for 160 – 280 bp and DNA eluted as described for GRO-seq.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

23544707

Reads aligned (%)

54.7

Duplicates removed (%)

20.7

Number of peaks

8538 (qval < 1E-05)

mm9

Number of total reads

23544707

Reads aligned (%)

54.6

Duplicates removed (%)

20.8

Number of peaks

8505 (qval < 1E-05)