K4 HP 3d: fat bodies from 3d bees fed control diet (honey and pollen)
Each RNA-Seq library (a total of 12 libraries were sequenced for the study: 2 ages x 2 diets x 3 replicate colonies) were prepared using Illumina’s TruSeq RNA Sample Preparation Kit according to the manufacturer’s protocols. Briefly, poly-A containing mRNA was purified using poly-T oligo-attached magnetic beads and was subsequently fragmented and primed for first-strand cDNA synthesis with random hexamer primers. After degrading the RNA template used for first-strand cDNA synthesis, synthesis of the second cDNA strand followed, yielding a double-stranded cDNA molecule. Adapters were ligated to both ends of the double-stranded cDNA molecules and adapter-ligated cDNAs were enriched using 15 cycles of PCR using adapter-specific primers. The libraries were validated according to the manufacturer’s protocol and the approximately 350 bp fragments were isolated from a 6% TBE PAGE gel and run through the Illumina sequencer for 2 x 100 cycles at a starting concentration of 12 pM per library.