Four intact high quality mid-gestational brains, two from 15–16 post-conceptual weeks (pcw) and two from 21pcw (Suppl. Table 1), were used to create detailed de novo reference atlases and transcriptome datasets (Fig. 1). The entire left hemisphere of each specimen was coronally, serially cryosectioned onto polyethylene naphthalate (PEN) membrane slides for laser microdissection (LMD), with interleaved slides for histological staining (Nissl, acetylcholinesterase (AChE), and in situ hybridization (ISH) for GAP43) for detailed structure identification. Approximately 300 anatomical regions per specimen were isolated for RNA isolation, amplification and microarray analysis on custom 64K Agilent microarrays23 (Fig. 1d; Suppl. Table 2; Extended Data Fig. 1). For one 16pcw and one 21pcw specimen, the right hemisphere was processed similarly but used for ISH (Fig. 1b) and Nissl staining. These data were anatomically delineated to make digital reference atlases (Fig. 1a), which allow the representation of transcriptome data in native anatomical coordinates. Figure 1e illustrates the specificity of anatomical sampling using this representation. For example, the folate receptor FOLR1 is selectively expressed in VZ and GE. Sufficient folate intake is essential for proper neuronal
