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

Ly6C High macrophages on Day 1 after muscle injury

cell type

Muscle infiltrating macrophages

time

Day 1 after muscle injury

Sequenced DNA Library

library_strategy

ATAC-seq

library_source

GENOMIC

library_selection

other

library_construction_protocol

Fascia of the injured TA muscle was removed. Muscles were dissociated in RPMI containing 0.2% collagenase B (Roche Diagnostics GmbH) at 37°C for 1 hour and filtered through a 100 µm and a 40 µm filter. CD45+ cells were isolated using magnetic sorting (Miltenyi Biotec). For FACS, macrophages were treated with Fcγ receptor blocking antibodies and with 10% normal rat serum: normal mouse serum 1:1 mix, then stained with a combination of PE-conjugated anti-Ly6C antibody (HK1.4, eBioscience), APC-conjugated F4/80 antibody (BM8, eBioscience) and FITC-conjugated Ly6G antibody (1A8, Biolegend). Ly6Chigh F4/80low macrophages, Ly6Clow F4/80high macrophages and Ly6Ghigh Ly6Cmed F4/80- neutrophils were quantified. In each experiment, compared samples were processed in parallel to minimize experimental variation. Cells were analyzed on a BD FACSAria III sorter and data analysis was performed using BD FACSDIVA and FlowJo V10 software. ATAC-seq was carried out as described earlier with minor modification (Buenrostro J.D. et al. 2013). 20 000 cells were sorted in ice-cold PBS. Nuclei were isolated with ATAC-Lysis Buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL) and were used for tagmentation using Nextera DNA Library Preparation Kit (Illumina) from 2-3 biological replicates. After tagmentation DNA was purified with MinElute PCR Purification Kit (Qiagen). Tagmented DNA was amplified with Kapa Hifi Hot Start Kit (Kapa Biosystems) using 9 PCR cycles. Amplified libraries were purified again with MinElute PCR Purification Kit. Fragment distribution of libraries was assessed with Agilent Bioanalyzer and libraries were sequenced on a HiSeq 2500 platform.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

19120619

Reads aligned (%)

96.7

Duplicates removed (%)

29.6

Number of peaks

51916 (qval < 1E-05)

mm9

Number of total reads

19120619

Reads aligned (%)

96.5

Duplicates removed (%)

29.7

Number of peaks

51858 (qval < 1E-05)