GSM8658452: Microglia WT replicate 3; Mus musculus; ATAC-seq
Brains were  dissociated using the Neural Tissue Dissociation kit (P) (Miltenyi Biotec), followed by removal of myelin and then MACS isolation of CD11b+ microglia following the manufacturer's guidelines (Miltenyi Biotec). Approximately 500,000 microglia were isolated from the brains of mice and centrifuged at 500 rcf for 10 minutes. Cells were resuspended in 47.5µl lysis buffer (10mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPA, CA-630 in water) at room temperature. After the addition of 2.5µl DNA enzyme I (Illumina), cells were incubated at 37°C for 30 min. DNA was purified with Zymo ChIP DNA concentrator columns (Zymo Research), eluted with 11µl of elution buffer, and amplified using NEBNext High-fidelity 2x PCR MasterMix (New England BioLabs) with the Nextera primer Ad1 (1.25µM) and a unique Ad2.n barcoding primer (1.25µM) for 8-10 cycles. Resulting libraries were size selected by gel excision to 165-250 bp, purified, and single end sequenced using a NovaSeq 6000 (Illumina) sequencer for 51 cycles according to the manufacturer's instructions.