GSM7157516: H3K27ac ChIP-seq for E10 brain cortex, rep2; Mus musculus; ChIP-Seq
Single cell suspension was prepared from E10 to E17 CD-1 mouse embryos. Four brains were collected for each time point. For E10 and E11 time points, ventricular zone was dissected from the tip of the brain. For E12 to E17 time points, brain tissues were first embedded in 4% low melting agarose at 35-37 °C and sectioned at 200 μm thickness with a vibratome (Leica) in ice-cold and oxygen-bubbling artificial cerebrospinal fluid (ACSF) containing: 125 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 25 mM NaHCO3, and 20 mM glucose (pH 7.4). The brain sections were transferred to a 3.5 cm dish containing ice-cold hibernate-E medium, and then ventricular zone was dissected and picked up under a stereoscope. The isolated VZ tissues were digested with papain using a commercial kit (Worthington, LK003150) at 37 °C for 30 min according to the manufacturer's recommendations. Single cell suspension was generated by dissociating the digested tissues through pipetting, washed with DPBS to remove digesting enzymes, and stained with DAPI in ice-cold DPBS. To enrich RGPs at later time points, FlashTag labeling was performed as previously described (Govindan et al., 2018). Briefly, pregnant mice were anesthetized, injected with FlashTag into the lateral ventricle of the uterine horns, and housed for 30 min. Then, single cell suspensions were prepared as described above. ChIP-seq libraries were prepared as described previously (Brind'Amour et al., 2015) with minor modifications: A total of 1 × 105 FlashTag+ cortical cells were FACS-purified and pelleted immediately after sorting for each experiment. The pelleted cells were frozen in liquid nitrogen and stored at -80 °C until further use. Cells were resuspended in cold Nuclear Isolation Buffer (Sigma-Aldrich, NUC-101). Before immunoprecipitation (IP), cells were thawed and digested using 80 U MNase (New England Biolabs, M0247S) for 5 min at 37 °C. The digestion was stopped by adding 10% of the reaction volume of 100 mM EDTA (Invitrogen, AM9260G). Chromatin was precleared in complete immunoprecipitation buffer with 10 µL Protein A/G Dynabeads (Invitrogen, 10001D and 10003D) for 2 hours at 4 °C with rotation. Meanwhile, 10 µL Protein A/G Dynabeads were mixed with 2 µL H3K27me3 antibody (Cell Signaling Technology, 9733) or H3K27ac antibody (Abcam, ab4729) under rotation for 3 hours at 4 °C in 100 µL complete immunoprecipitation buffer. The precleared chromatin was mixed with the antibody-bound beads under overnight rotation at 4 °C. Beads were then washed twice with low salt wash buffer and twice with high salt wash buffer. DNA was then eluted for 1.5 hours at 65 °C in 30 µL ChIP elution buffer. DNA was purified using 2.2 × VAHTS DNA Clean Beads and eluted in ultrapure H2O (Thermo Fisher Scientific, 10977015). For H3K27ac IP, 10 mM sodium butyrate (Sigma-Aldrich, 303410) was added in cold Nuclear Isolation Buffer, complete immunoprecipitation buffer, low salt wash buffer, and high salt wash buffer. Libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit (New England Biolabs, E7645S), according to the manufacturer's instructions. Libraries were PCR amplified for 13-15 cycles using NEBNext® Multiplex Oligos (New England Biolabs, E7335S). The amplified libraries were purified using 2.2 × VAHTS DNA Clean Beads and eluted in ultrapure H2O.