Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Heads from 50 gynes, 50 large workers or 200 small workers originating from the same colony were pooled and used for RNA extraction as described previously (Yek et al. 2013). The remaining body parts were likewise pooled per caste and colony and subsequently used for DNA extraction. Genomic DNA was extracted using a Blood & Cell Culture Midi kit (Qiagen) following the instructions enclosed in the kit, with the modification that sample lysis was performed overnight. For DNA samples, 5 μg genomic DNA per sample was fragmented by sonication with a Covaris S2 system (Covaris, MA) to a mean size of approximately 500 bp, followed by end-repair, 3'-end addition of dA, and adapter ligation. The ligated fragments were size selected at 500 bp on an agarose gel and amplified by 10 cycles of PCR to yield the DNA libraries. All libraries were subjected to 90 bp pair-end sequencing on an Illumina HiSeq 2000 platform. For RNA samples, strand-specific RNA-Seq libraries were constructed according to Parkhomchuk D, et al (2009). Briefly, 2 μg total RNA per sample was first treated with DNase I (New England BioLabs) to remove the possible contamination of genomic DNA. Then poly (A+) mRNA was purified from the total RNA using the Dynabeads mRNA Purification Kit (Invitrogen), followed by fragmentation using the RNA fragmentation kit (Ambion). Next, the first cDNA strand was synthesized using random hexamer primers and reverse transcriptase (Invitrogen), and second-strand cDNA was synthesized using DNA polymerase I (New England BioLabs) where dUTP was used instead of dTTP. After that, the synthetic double-strand cDNA was end repaired, followed by 3'-end addition of dA, adapter ligation and size selection according to Illumina's protocols. Finally, the selected products were treated by UNG (Applied Biosystems) and amplified by 15 cycles of PCR to yield the strand-specific RNA-Seq libraries. All the libraries were subjected to 90 bp pair-end sequencing on the Illumina HiSeq 2000 platform.