Honey Bee Forager Brain RNA-Seq
RNA was purified, DNAse treated, and eluted with 30 ul of RNAse free H20, using the RNeasy Mini Kit and the RNase-Free DNase Set (Qiagen, Valencia, CA, USA). 50 ng of pooled RNA was reverse transcribed with oligo-d(T) and random primers into cDNA, and linearly amplified using NuGEN’s Ovation RNA-Seq protocol (NuGEN, San Carlos, CA, USA). Samples were then fragmented (Covaris, Woburn, MA, USA), end-repaired, d-A tailed, and adaptor-ligated with the NEBNext DNA Sample Prep Reagent set (New England Biolabs [NEB], Ipswich, MA, USA). The ligation product was then run on a 2% TAE agarose gel to excise an approximately 300 bp fragment. Enrichment PCR (15 cycles) was performed using Phusion High-Fidelity PCR Master Mix (NEB). Libraries were then quantified on a bioanalyzer with a High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA, USA). Whole transcriptome paired-end data was generated with the HiSeq 2000 (Illumina, San Diego, CA).