HiSeq2000 sequencing for pool of 8 worker honeybee brains (3 of 5)
Libraries of queen/worker and reverted nurse/forager samples were created using TruSeq DNA library preparation kits (Illumina) with some modification to the standard protocol. Genomic DNA samples were prepared by homogenizing pools of whole brains. For queen/worker samples, 5 pools of 8 brains per pool were prepared. Genomic DNA was extracted from homogenized brains using either Masterpure kit from Epicentre (queen/worker), or lysing cells in Chaos buffer (4.5M Guanidinium thiocyanate, 2% N- lauroylsarcosine, 50mM EDTA, 25mM Tris-HCl, 0.1M b-Mercaptoethanol) and purified with Qiagen DNeasy columns (reverted nurse/forager). For all samples, Genomic DNA was sheared to an average size of 350 bp using a Covaris sonicator with the following settings: Duty cycle = 10%, Intensity = 5.0, Bursts per second = 200, Duration = 60 sec. Blunt ends were created on the DNA fragments using a unique protocol to eliminate the introduction of non-genomic cytosines into the fragments, which would be falsely interpreted as unmethylated cytosines during subsequent analysis. To achieve this, we only used A, G and T nucleotides with a mixture of the enzymes T4 DNA polymerase, Klenow DNA polymerase and T4 PNK(NEB) to perform end repair of fragments. Illumina adapters were then ligated to the fragments after the addition of a single A, per TruSeq protocol. Libraries were then size selected by cutting a 400-500 bp fragment from an agraose gel (Bio-Rad-Certified Low Range Ultra Agarose, NEB-100 bp DNA Ladder, Invitrogen-SYBR® Gold nucleic acid gel stain) and purified using Qiagen MinElute Gel Extraction Kit. The purified libraries were then bisulfite converted and purified using Zymo EZ DNA Methylation Gold. Libraries were then amplified using a mixture of Uracil insensitive polymerases; Denville Choice Taq and Agilent Turbo Pfu. Queen/worker samples were amplified for 12 cycles and reverted nurse/forager for 15 cycles using the TruSeq PCR conditions.