GSM7157509: H3K27me3 CUT&Tag for E14-E15 brain cortex, rep1; Mus musculus; OTHER
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. A total of 1 × 105 FlashTag+ cortical cells were FACS-purified and pelleted immediately after sorting for each experiment. CUT&Tag libraries were prepared using NovoNGS® CUT&Tag® 3.0 High-Sensitivity Kit (Novoprotein, N259-YH01), according to the manufacturer's instructions.