GSM8574832: BV2_Ctrl_hyp_rep2_ATACseq; Mus musculus; ATAC-seq
ATACseq was performed as previously described with some modifications (Chen et al., 2016). While still in hypoxic conditions, cells were fixed with 1% formaldehyde (Thermo Scientific) for 10 min and then quenched with 0.125 M glycine for 5 min at room temperature. After returning to normal conditions (21% O2), cells were washed with PBS and incubate in 3% BSA (Sigma) with CD16/CD32 Fc Block™ (1:250, BD Pharmingen, Cat#553142) for 15 min. Next the CD45 (Clone 30-F11) labeled with PE, (1:400, BD Pharmingen, Cat#553081) was added for 40 min, washed with PBS, and CD45+ cells or GFP+ cells were sorted to 1% BSA in PBS using Cell Sorter BD FACSAriaII. After sort, cells were counted and 50,000 cells lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) for 5 min on ice. Cells were then centrifuged at 500xg for 8min, and cell pellets were resuspended in transposition reaction according to the standard ATACseq protocol using 2.5 ul of Tn5 enzyme per 50 000 of cells (Buenrostro et al., 2015). After transposition, cells were centrifuged and reverse crosslinked (50 mM Tris-Cl, 1 mM EDTA, 1% SDS, 0.2 M NaCl, 200 μg/ml proteinase K) at 65°C with an overnight shaking at 1200 rpm. Zymo DNA Clean & Concentrator-5 columns (ZymoResearch) were used to purify DNA. Sequencing libraries were prepared according to the original ATACseq protocol (Buenrostro et al., 2015). The ATACseq libraries were assessed for appropriate quality using a Bioanalyzer 2100 and subjected to paired-end sequencing (2x151bp) using NovaSeq 6000 (Illumina). Two biological replicates per condition were analysed.