title: GSM9514005: ATAC-seq of mouse microglia, MCAO day 1, wild-type; Mus musculus; ATAC-seq
library_construction_protocol: Sham-operated mice or mice after MCAO were transcardially perfused with saline, and then the forebrain was removed. After homogenization with RPMI-1640, 1 mg ml–1 collagenase (Sigma-Aldrich) and 50 mg ml–1 DNase I (Sigma-Aldrich) were added to the homogenates. Brain homogenates were digested for 25 min and subjected to Percoll (GE Healthcare) gradient centrifugation. From the interlayer between 37% and 70% Percoll, cells were isolated and stained with anti-CD45 FITC (30-F11, eBioscience), anti-CD11b PerCP-Cy5.5 (M1/70, eBioscience), and anti-CD31 PE/Cyanine7 (390, eBioscience). The cells were sorted using a FACSAria III cell sorter (BD/Thermo Fisher Scientific). An ATAC-seq library was prepared according to the protocol described previously (Nakamura et al., Neuron 2023). For the fragmentation and tagmentation of genomic DNA, 50,000 nuclei isolated from CD45(int)CD11b(int) cells were treated with Tn5 transposase (Illumina, Nextera DNA Library Preparation Kit). The DNA fragments were amplified using KAPA HiFi HS ReadyMix (Kapa Biosystems) and purified using SPRI select beads (Beckman). Library quality and fragment size distribution were assessed by Agilent TapeStation 4150. Libraries were sequenced using the Illumina HiSeq 2500 for wild-type time-course samples or Illumina NovaSeq 6000 for Igf1-CreER;Ai14 samples.
identifiers.namespace: GEO
identifiers.id: GSM9514005_r1