RNA-seq of Apis mellifera: common nurses from common colonies
Three g of RNA per honey bee sample was used as input material for RNA sample preparations. First strand cDNA was obtained using random hexamer primer and M-MuLV Reverse Transcriptase, second strand cDNA was obtained using DNA Polymerase I and RNase H. DNTPs with dTTP were replaced by dUTP in the reaction buffer. Remaining overhangs were converted into blunt ends by exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. AMPure XP system (Beckman Coulter, Beverly, USA) was used to purify the library fragments to select cDNA fragments of preferentially 150-200 bp in length. Lastly, products were purified (AMPure XP system) and library quality was assessed with the Agilent Bioanalyzer 2100 system. The libraries were sequenced on anIllumina Hiseq 2500 platform and 100 bp paired-end reads were generated.