title: GSM9514201: CUT&Tag anti-YY1 of mouse microglia, MCAO day 28, Cx3cr1-CreER;Zfp384flox/flox; Mus musculus; OTHER
library_construction_protocol: For primary microglia: 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). CD45(int)CD11b(int) cells were sorted using a FACSAria III cell sorter (BD/Thermo Fisher Scientific). For BV2 cells: BV2 cells were harvested from culture. CUT&Tag libraries were prepared using a bench top CUT&Tag V.2 protocol with slight modifications, as described previously (Nakamura et al., Neuron 2023). BV2 cells or CD45(int)CD11b(int) cells (100,000–200,000 cells from the sham-operated or ischemic hemisphere) were pelleted for 3 min at 600×g at room temperature. Anti-YY1 (Abcam, ab109237) and anti-ZNF384 (Sigma, HPA004051) antibodies were used for primary antibodies. Sequencing was performed using the PE150 strategy on Illumina HiSeq 2500 for primary microglia and BV2 IgG samples, or Illumina NovaSeq 6000 for BV2 anti-ZFP384 and anti-YY1 antibody samples.
identifiers.namespace: GEO
identifiers.id: GSM9514201_r1