GSM8994471: EB2-2 flox mouse; Mus musculus; RNA-Seq
Two mice from EB2-flox and EB2-vGATCre group respectively were anesthetized with isoflurane and rapidly decapitated. Brains were extracted and transferred into ice-cold Hibernate A/B27 medium. The PFC was removed from slices and isolated intact nucleus suspension. The nucleus was isolated and purified as previously described with some modifications. Briefly, the frozen tissue was homogenized in NLB buffer containing 25mM Sucrose, 10 mM Tris-HCl, 3 mM MgAc2，0.1% Triton X-100 (Sigma-Aldrich), 0.1mM EDTA, 0.2 U/μL RNase Inhibitor(Takara). Various concentrations of sucrose were used to purify the nuclei. Nuclei counts were determined by PI staining. Finally, the concentration was adjusted to 1000 nuclei/μL for snRNA-Seq. Single-nucleus RNA-seq libraries were constructed using the 10X Genomics Chromium Controller and Chromium Single Cell 3' v3 Reagent Kit according to the manufacturer's standard protocol. Briefly, nuclei were isolated, loaded into microfluidic chips to generate Gel Bead-In-Emulsions (GEMs), followed by reverse transcription, cDNA amplification, fragmentation, A-tailing, adaptor ligation, and index PCR. Final libraries were quantified using Qubit and size-checked with a Bioanalyzer.