GSM3244434: RNA-Seq drone head (experiment 1); Apis mellifera; RNA-Seq
Total RNA samples were isolated using the RNeasy mini kit (QIAGEN #74106) including on-column DNase treatment. mRNA was purified from 10–50 μg of total RNA by two cycles of poly-A enrichment using the Oligotex kit (QIAGEN #72022) followed by an rRNA removal step using the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen #A1083702). Precipitated mRNA samples were eluted with 9 ml of RNase free water and fragmented with 1 ml of fragmentation buffer (Ambion, #AM8740) at 70 C. Reactions were stopped after 5 min by adding 1 ml stop buffer, and RNA was purified by ethanol precipitation. cDNA was synthesized from 100–300 ng of mRNA using SuperScript III reverse transcriptase (Invitrogen #18080-051). Double-stranded DNA was synthesized according to the instructions using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). DNA was cleaned with a QIAquick PCR spin column (QIAGEN, #28106), sequencing adapters were ligated according to the Illumina protocol, and the library was amplified by 18 cycles of PCR using Phusion DNA polymerase (NEB, #F-530). Bands around 300 bp were gel purified and libraries were sequenced on the Illumina HiSeq 2000 platform at the Vincent J. Coates Genomic Sequencing Laboratory at UC Berkeley to generate single-end 100 base reads. Honey bee samples were collected in two rounds (for two biological replicates). Worker embryos (eggs) were transferred from honeycomb cells into microcentrifuge tubes (50 per/tube). Drone and worker larvae were obtained at about 3rd and 4th instar developmental stages. Worker pupae were collected at about pink eyes developmental stage. Worker heads were collected from bees newly emerged from their own growing cells. Drone heads were collected from the same colony as drone larvae and worker embryos, larvae, pupae, and adult heads were collected from. Semen was collected from mature drones essentially as described in (Robinson et al., 2000). All samples were flash frozen in liquid nitrogen immediately upon collection. Except for queen heads, for which we used a single head per experiment, the rest of the biological sampling consisted of a mix of individuals, three larvae/pupae/heads, 50 embryos, and 20ul of semen. RNA-Seq: Total RNA samples were isolated using the RNeasy mini kit (QIAGEN #74106) including on-column DNase treatment. mRNA was purified from 10–50 mg of total RNA by two cycles of poly-A enrichment using the Oligotex kit (QIAGEN #72022) followed by an rRNA removal step using the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen #A1083702). Precipitated mRNA samples were eluted with 9 ml of RNase free water and fragmented with 1 ml of fragmentation buffer (Ambion, #AM8740) at 70 C. Reactions were stopped after 5 min by adding 1 ml stop buffer, and RNA was purified by ethanol precipitation. cDNA was synthesized from 100–300 ng of mRNA using SuperScript III reverse transcriptase (Invitrogen #18080-051). Double-stranded DNA was synthesized according to the instructions using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). DNA was cleaned with a QIAquick PCR spin column (QIAGEN, #28106), sequencing adapters were ligated according to the Illumina protocol, and the library was amplified by 18 cycles of PCR using Phusion DNA polymerase (NEB, #F-530). Bands around 300 bp were gel purified and libraries were sequenced on the Illumina HiSeq 2000 platform at the Vincent J. Coates Genomic Sequencing Laboratory at UC Berkeley to generate single-end 100 base reads.
GEO Accession
GSM3244434