Validation of RNA-seq by quantitative RT-PCR and Confirmation of W-linkage. (A) Blastoderm expression analysis of four representative W genes, KCMF-W, RASA-W, MIER3-W and ZNF532. Expression was detectable in females (red) but not in males (blue). Normalised W gene expression is shown; mean +/- SEM; n = 3; ** P <0.05. (B-E) FISH mapping of genes identified by RNA-seq to the W sex chromosome in female chicken metaphase spreads. BAC clones were used as probes. (B) BAC clone Ch261-113E6 (ZNR-W, BTF3-W) (red) and BAC Ch261-178N8 (RASA1-W, BTF3-W) (green). (C) BAC clone Ch261-107E4 (HNRPK2-W, GOLPH3-W (red). (D) BAC clone Ch261-60P24 (ZNF532-W, SnoR58-W) (red). (E) BAC clone Ch261-114G22 (UBE2R2-W, RASA1-W, SnoR121A-W) (red). Metaphase chromosomes are stained with DAPI (blue). A single signal was detected in each case and only in female cells, confirming W linkage.