title: GSM3712329: Wild-type, replicate 2, Ly6C Low macrophages on Day 2 after muscle injury.; Mus musculus; ATAC-seq
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.
attributes.tag: GEO Accession
attributes.value: GSM3712329