GSM9018436: RNA_E6.5_EXE_EED_DMSO_2; Mus musculus; RNA-Seq
For RNA-seq, intact or isolated embryos were collected and immediate freeze at -80 °C until use. SMART-Seq™ v4 Kit (Clontech, 634890) was used to constructed cDNA libraries.  For CUT&RUN, intact or isolated embryos were mixed with activated Concanavalin A Magnetic Beads (Polysciences, 86057-3) for 10 mins at RT, followed the incubation with primary antibodies overnight at 4°C. After three washes, samples were included with 3 ng/μl pA-MNase (home-made) for 2 hours with rotation at 4 °C. Subsequently, pA-MNase was activated by incubating with 200 μl pre-cooled 0.5 μM CaCl2 for 20 mins at 4 °C, and quench by adding 23 μl 10×stop buffer. To release DNA, samples were first incubated at 37 °C for 15 minutes, followed by addition 2.5 μl 10% SDS and 2.5 μl Proteinase K (20 mg/ml; Thermo Fisher, AM2546), and then incubated at 55 °C for 1 hour. DNA was subsequently purified using phenol-chloroform extraction.  For ATAC-seq, isolated embryos were incubated with Tn5 (Diagenode, C01070012-30) for 15 mins at 37°C. Subsequently, DNA fragments were released by dissecting with Proteinase overnight at 55°C. Nextera® XT DNA Sample Preparation Kit kit (Illumina, FC-131-1024) was used for the RNA-seq libraries construction. For CUT&RUN, libraries were constructed using NEBNext Ultra II DNA library preparation kit (New England Biolabs, E7645S). ATAC-seq ibraries were constructed with NEBNext High-Fidelity 2×PCR Master Mix (NEB, M0541S).