GSM8181858: Wild-type, replicate 1, Ly6C High macrophages on Day 1 after muscle injury, ChIP-seq; Mus musculus; ChIP-Seq
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). Cells were isolated for downstream applications on a BD FACSAria III sorter. The monocyte purification procedure was performed as described previously (Houthuys et al., 2010) with slight modifications. In short, the blood from C57BL/6 mice was taken by cardiac puncture to maximize the amount of blood obtained in an endotoxin-free manner. Up to 5.3 × 10^7 ± 4 × 10^6 white blood cells (viability >96%) were obtained from 15 ml of blood from 15 animals after red blood cell lysis (n = 3). To minimize cell aggregation and adhesion to plastic, all purifications were performed at 0–4°C in PBS/BSA/EDTA (MACS buffer). For red blood cell lysis, cells were resuspended in 0.5–1 ml FACS Lysing solution (BD; Cat #349202), incubated for 5 min at room temperature, and then centrifuged (400 g, 5 min, 4°C). For FACS and sorting, MFs were treated with Fcγ receptor blocking antibodies, 5% normal rat serum, and 5% normal mouse serum. They were then stained with a combination of APC-conjugated anti-Ly6C antibody (eBioscience; HK1.4), PE/Cy7-conjugated CD11b antibody (BD Bioscience; M1/70), PE-conjugated Ly6G antibody (BioLegend; 1A8), and PerCP/Cy5.5-conjugated MHCII antibody (BioLegend; M5/114.15.2). Gating and sorting strategy are shown in Patsalos et al. JEM 2022. 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.