2000 by the Beijing Genomics Institution (BGI, Beijing, China). To analyze RNA-Seq data, tophat2 was used for reads mapping to the mm10 reference and cufflinks to estimate the FPKM. To identify differentially expressed genes, we employed cuffdiff and drew heatmaps using R gplots package. Differentially expressed transcripts were defined by FDR cuffdiff of 5% and fold changes 2. We performed KEGG and gene ontology (GO) enrichment ≤ ≥ analysis using R GO stats package and visualized for pathways with an R pathview package. The obtained transcriptome metadata was deposited as the accession number: SRA320651 in Sequence Read Archive (SRA) database of National Center for Biotechnology Information (NCBI). Rescue assay analysis Littermate control and DTG male mice were treated with Dox from P18, and depilated at P21. Hh pathway activation was measured as previously described with some modification (). Briefly, littermate control mice were treated with a single 25 L topical application of vehicle (95% acetone/5% DMSO) across depilated dorsal skin. DTG mice were treated with a single 25 L topical application of SAG (0.06 g per L in vehicle, 120 mM). Second dose was applied 24 hours later. Hh pathway activation was assessed by studying Gli1 on Western blotting. hybridization In situ hybridizations for were performed as the previously described (; ). In situ Shh Construct used to generate probe was a gift from Maksim V. Plikus (UC Irvine). Shh In situ probe and anti-sense probe were generated by DIG RNA Labeling Kit (SP6/T7) Shh according to the manufacturer’s instructions (Roche, Mannheim, Germany) and purified by illustra ProbeQuant G-50 Micro Columns kit (GE Healthcare, Pittsburgh, PA). Double probe and anti-sense probe were hybridized at 65°C. To detect the signal, slides Shh were stained with anti-Digoxigenin-AP antibody (Roche, Mannheim, Germany) and color
2000 by the Beijing Genomics Institution (BGI, Beijing, China). To analyze RNA-Seq data, tophat2 was used for reads mapping to the mm10 reference and cufflinks to estimate the FPKM. To identify differentially expressed genes, we employed cuffdiff and drew heatmaps using R gplots package. Differentially expressed transcripts were defined by FDR cuffdiff of ≤ 5% and fold changes ≥ 2. We performed KEGG and gene ontology (GO) enrichment analysis using R GO stats package and visualized for pathways with an R pathview package. The obtained transcriptome metadata was deposited as the accession number: SRA320651 in Sequence Read Archive (SRA) database of National Center for Biotechnology Information (NCBI). Rescue assay analysis Littermate control and DTG male mice were treated with Dox from P18, and depilated at P21. Hh pathway activation was measured as previously described with some modification (). Briefly, littermate control mice were treated with a single 25 L topical application of vehicle (95% acetone/5% DMSO) across depilated dorsal skin. DTG mice were treated with a single 25 L topical application of SAG (0.06 g per L in vehicle, 120 mM). Second dose was applied 24 hours later. Hh pathway activation was assessed by studying Gli1 on Western blotting. In situ hybridization In situ hybridizations for Shh were performed as the previously described (; ). Construct used to generate Shh In situ probe was a gift from Maksim V. Plikus (UC Irvine). Shh probe and anti-sense probe were generated by DIG RNA Labeling Kit (SP6/T7) according to the manufacturer’s instructions (Roche, Mannheim, Germany) and purified by illustra ProbeQuant G-50 Micro Columns kit (GE Healthcare, Pittsburgh, PA). Double Shh probe and anti-sense probe were hybridized at 65°C. To detect the signal, slides were stained with anti-Digoxigenin-AP antibody (Roche, Mannheim, Germany) and color