title: RNA-seq analysis of floral volatile gene expression in Cypripedium henryi in response to pollination treatments
design: RNA-seq analysis of floral volatile gene expression in Cypripedium henryi in response to pollination treatments
library_construction_protocol: Sampling took place in the greenhouse 48 h (± 2 h) after pollination treatments. The labellum, staminode, and both lateral petals of each treated flower were collected using a thin razor blade and immediately flash-frozen in liquid nitrogen for transport to the laboratory. Two multiflowered clonal individuals of Cypripedium henryi were maintained year-round according to species requirements under the care of expert Cypripedium horticulturalists. Three biological replicates were used for each of the four treatments (n=12 flowers total). To minimize potential bias, replicates were distributed evenly across the two clonal individuals. The experimental treatments were executed as follows: • Treatment P (Pollen Removal): Manual removal of both pollinia.   • Treatment G (Cross-Pollination): Deposition of pollinia from a donor flower onto the stigma.   • Treatment GP (Combined): Manual removal of both pollinia followed by the deposition of donor pollinia onto the stigma.   • Treatment K (Control): Untouched flowers served as the negative control.  The whole staminode (around 15 mg), half of the labellum (around 60 mg) and a whole lateral petal (around 30 mg) were used for total RNA extraction using the protocol of innuPREP Plant RNA Kit (Innuscreen GmbH). Prior to the extraction, the tissue samples were crushed using a mechanical mortar. Total RNA was eluted in 20 μl RNAse free H2O. Total RNA concentration and purity were determined by RNA screen tape analysis the Tapestation (Agilent Genomic Screentape, Agilent Technologies) performed on the Genomic Screen Tape device (Agilent Technologies) according to the manufacturer's instructions. RIN number of samples ranged between 5.8 and 8.4. Strand-specific RNA-seq libraries were prepared by BMKGENE (Münster, Germany). Following poly(A) enrichment of the mRNA fraction, libraries were constructed using the dUTP method.