CUT&Tag Seq for H3K27ac of mus musculus: E9.5 embryo neural tissues affected with neural tube defects
Pregnant mice were given BaP (CAS-No. 192-97-2, purity 98%, Sigma, USA) intraperitoneally, dissolved in corn oil, from E6.5 for three consecutive days at a dose of 250 mg kg-1. On E9.5, embryos were collected and carefully inspected for NTDs under a dissecting microscope. Ten embryos were pooled as a sample for CUT&Tag experiment. Embryonic neural tissues, from the most rostral aspect of the forebrain to the caudal aspect of the hindbrain, were excised. Neural tissues were homogenized and followed by incubation with Concanavalin A-coated Magnetic Beads (Novoprotein, China) for 10 min at room temperature. Next, H3K27ac rabbit pAb (1:100, Merck Millipore, USA) were employed as primary antibodies, and Goat Anti-Rabbit IgG H&L (1:100, Abcam, USA) was used to bind primary antibodies. After binding pG-Tn5 adapter complex (Novoprotein, China), tagmentation was conducted. Following library amplification, DNA quantity was determined with Qubit 4 (Thermo Fisher Scientific, USA) and library quality characterized with Agilent TapeStation 4200. All libraries were sequenced using NovaSeq 6000 (Illumina, USA).