WJ-John-586_TAP_S1
Apis mellifera
Protocols: Royal jelly was collected from queen brood cells containing 3 days old larvae, and worker jelly was collected from 5 days old worker larvae brood cells; all brood belonged to untreated healthy looking hives. The jelly samples were immediately frozen in liquid nitrogen and kept at -80°C for further analysis. Total RNA extracted from worker and royal jellies was subjected to Tobacco Acid Pyrophosphatase (TAP) treatment using the Cap-Clip enzyme (CellScript). Modified RNA was purified again by standard Phenol/Chloroform extraction followed by Ethanol precipitation in the presence of Glycogen. The RNA pellet was taken up in 12 ul nuclease-free water. RNA quality and quantity was verified using Agilent RNA 6000 pico chip on a Bioanalyzer 2100 instrument. Full-length RNA libraries were prepared using the NEXTflex small RNA-seq kit v3 (Bioo Scientific) according to the manufacturer's instructions with the following modifications; Adaptor ligation was performed at 16°C overnight (step A). The bead cleanup (step F) was performed following an amended one-sided purification protocol to retain also long fragments (no size selection protocol) as provided by the manufacturer. The final purification of the PCR product (step H1) followed also the amended protocol without size selection as provided by the manufacturer.