GSM9465533: ATAC-seq in Egr2 knock-out alveolar macrophages (plated, 24h IL-4-treated, rep1); Mus musculus; ATAC-seq
The murine alveolar macrophages were isolated by bronchoalveolar lavage (BAL) from Egr2+/+ and Egr2fl/fl mice. To collect BAL samples, animals were euthanized by isoflurane inhalation and their tracheas were cannulated. Lavage was performed with 2 aliquots of 0.7 ml of ice-cold PBS (pH 7.3). The BAL cells were centrifuged at 400 g for 10 minutes at 4 °C and the supernatants were removed. BAL cells were suspended in ACK buffer (4.012 g NH4Cl, 0.5 g KHCO3, 18.61 mg Na2EDTA x 2H2O, and 0.5 L milliQ; pH 7.2-7.4) to lyse red blood cells for 2 minutes at room temperature and then adding 1 mL of MACS buffer (2 mM EDTA and 0.5 % BSA in 1x PBS (pH: 7.2)). The alveolar macrophages were purified with CD45 magnetic bead selection according to the manufacturer's protocol (CD45 bead isolation kit; Order No. 130-052-301; Miltenyi). We cultured isolated cells at 24-well plates in 0.5 mL RPMI medium containing 1% penicillin/streptomycin/amphotericin B (A5955; Sigma), and 10% FBS (F7524, Sigma). After attachment, we added 20 ng/ml IL-4 (Peprotech; 214-14). ATAC-seq was performed as previously (Kolostyak et al. JCI Insight (2024). Libraries were prepared from 10ng DNA withTruSeq ChIP library systems (Illumina) according to the manufacturer's instructions.