For deep layer neurons, mice expressing the Golli-GFP transgene were used. For upper layer neurons, a birth-dating plasmid expressing Cdk5r promoter-GFP was introduced into the neocortex by in utero electroporation at E15.5, during the peak of upper layer neurogenesis. Neocortex was micro-dissected from the embryonic brain and kept on ice. A total of 2-8 embryos were used per replicate. Cells were dissociated in papain, and GFP-positive cells were purified using FACS. 2.5 x 10^5 - 5 x 10^5 cells per sample were sorted into micro-centrifuge tubes containing neurobasal/DMEM-based cell culture medium and kept cold. Cells were then washed once in PBS and resuspended in cold lysis buffer. Nuclei were then pelleted by centrifugation for 10 minutes at 4 degrees C. Nuclei were washed again in PBS, then incubated in Tn5 transposition reaction mix for 30 minutes at 37 degrees C. DNA was purified using a Qiagen MinElute kit. ATACseq libraries were prepared for sequencing using standard protocols (Buenrostro et al. 2013). Briefly, transposed and purified DNA was amplified over 12 cycles of PCR using NEBNext High Fidelity PCR Master Mix (New England Biolabs M0451S). Libraries were then purified using a Qiagen PCR purification kit, and residual primers were removed using AMPure XP Beads (Beckman Coulter/Fisher NC9959336).