a, Samples for Geo-seq: embryonic stages, biological replicates and the germ layers that were sampled for Geo-seq analysis. Stages: E5.5–E6.0, pre-gastrulation stage; E6.5, early streak; E7.0, late mid-streak; E7.5, no-bud stage. See Supplementary Table 1 for complete information. b, The strategy of sampling of cell populations in the epiblast–ectoderm and endoderm from E5.5 to E7.5 embryos and mesoderm in the E7.0 to E7.5 embryos (outlined in c). Samples were designated by the sequential (ascending) order of serial sections (1 for the most distal section) and the regions in the section (R, reference section; S, sample section; see histology images for orientation). For E5.5 embryo, the anterior–posterior axis has not yet been determined, so samples were denoted as Epi 1/2 and En 1/2. For E5.5–E7.5 embryos: Epi, epiblast; En, endoderm; A, anterior; P, posterior; AP, pooled from anterior and posterior; L, left side (1, anterior; 2, posterior); R, right side (1, anterior; 2, posterior); embryonic axes: anterior–posterior, A↔P; proximal–distal, P↔D; left–right L↔R. Scale bar, 50 µm. d, Box plot showing the number of detected genes (FPKM > 0) for samples of E5.5 to E7.5 embryos (E5.5: n = 10; E6.0: n = 18; E6.5: n = 30; E7.0: n = 73; E7.5: n = 83). The centre line marks the median and box edges represent 25th and 75th percentiles. e, Gene-expression density plot of Geo-seq data of samples from E5.5 to E7.5 embryo; the solid line is the mean value and shaded region represents the s.d. f, Corn plots showing the expression of representative germ-layer markers (ectoderm: Sox2; endoderm: Sox17; mesoderm: Mesp1; visceral endoderm: Hnf4a) at E5.5 to E7.5 stages. Similar results were obtained from embryo replicates. g, The expression of anterior visceral endoderm (AVE) and distal visceral endoderm (DVE) markers (Cer1, Dkk1 and Lefty1) at E5.5 and E6.0 stages. Similar results were obtained from embryo replicates. h, The comparison of digitally rendered gene-expression pattern and WISH results of seven examples of genes in the epiblast and endoderm at E6.5 stage. WISH images were obtained from the EMAP eMouse Atlas Project (www.emouseatlas.org)56. Embryos are oriented with posterior to the right and distal to the bottom. i, The comparison of the expression of selected coding and noncoding RNAs by Geo-seq and WISH in E6.5 epiblast (Crb3, Car2), E7.0 (Gm37335, RP23-458G12.2) and E7.5 ectoderm (Hoxb3os, A930024E05Rik). n > 3 for each gene. Spearman correlation plots of WISH and Geo-seq results are shown on the right. Embryos are oriented with posterior to the right and distal to the bottom.