GSM9514204: H3K27ac HiChIP of mouse microglia, MCAO Day 6; Mus musculus; OTHER
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). CD45(int)CD11b(int) cells (200,000–300,000 cells from the sham-operated or ischemic hemisphere) were analyzed by HiChIP according to the protocol described elsewhere. After cells were sonicated for 2 min using a Covaris S220 ultrasonicator (Covaris), 2 µg of antibody against H3K27ac (Abcam, ab4729) and 20 µl of protein A beads (Thermo Fisher) were added to capture the chromatin-antibody complex. Captured chromatin was removed from protein A beads and purified by DNA Clean & Concentrator (Zymo, D4013) to be quantified by Qubit 3.0 (Thermo Fisher). After treatment with Tn5 transposase (Illumina, Nextera DNA Library Preparation Kit) for fragmentation and tagging, the library was prepared by amplification PCR (14 cycles) followed by the purification using SPRI select. Libraries were sequenced on Illumina HiSeq 4000.