Microglia ATAC-seq library generation

ATAC-sequencing libraries were generated using Nextera^® DNA Sample Preparation Kit (Illumina, FC-121-1030) following the methods described by [66 (link), 67 ]. A total number of 80,000 microglia were pooled from two animals (40,000 cells from each) and collected in Eppendorf tubes containing 300 µL medium A. Cells were pelleted by centrifugation (10 min, 4 °C, 500×g), resuspended in 50 μL of cold lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630) and immediately centrifuged as before. Next, nuclei were resuspended in 50 μL transposition reaction mix (1 × TD reaction buffer, 2.5 μL TN5 transposase) and incubated at 37 °C for 30 min. Immediately following transposition, the DNA was purified using a minElute PCR purification kit (Qiagen, 28004) following the manufacturer’s instructions. The transposed DNA fragments were further amplified and barcoded [66 (link), 67 ] and purified with a ChIP DNA Clean and Concentrator kit (Zymo, D5205). The fragments were run on 2% E-Gel™ EX agarose gels (Thermo Fisher scientific, G521802) and 150–600 bp fragments were excised, followed by purification with Zymoclean™ Gel DNA Recovery Kit (Zymo, D4007). Library concentration was determined with an Agilent 2100 Bioanalyzer after which 8 samples were pooled and sequenced using HiSeq Rapid SBS Kit v2 (50 cycles) using paired end reads on a HiSeq2500 (Illumina).