GSM8365374: LL_3_4; Mus musculus; ATAC-seq
ATAC-sequencing libraries were generated using Nextera® DNA Sample Preparation Kit (Illumina, FC-121-1030) following methods previously described (Buenrostro et al., 2015, 2013). A total number of 80,000 microglia were pooled from two animals (40,000 cells from each) and collected in siliconized microcentrifuge tubes (Sigma, T3406-250EA) containing 300 µL medium A. For each group, at least two pools of two mice each were processed. 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) (Illumina, 20034197) 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 (Buenrostro et al., 2015, 2013) 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 150–550 bp fragments were excised, followed by purification with Zymoclean™ Gel DNA Recovery Kit (Zymo, D4007). Library concentration was determined with Qubit dsDNA HS kit (Thermo Fisher Scientific, Q33230) and confirmed with Agilent 4200 TapeStation System, after which the 13 samples were pooled and sequenced using NextSeq 500/550 Kit v2.5 (92 cycles) using paired-end reads on a NextSeq500 (Illumina).