GSM3772905: BS-seq drone sperm (experiment 2); Apis mellifera; Bisulfite-Seq
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. About 500 ng of genomic DNA was isolated from various honey bee developmental stages, fragmented by sonication, end repaired, and ligated to custom synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer's (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (QIAGEN), as outlined in the manufacturer's instructions. The bisulfite-converted libraries were then amplified by PCR using ExTaq DNA polymerase (Takara Bio) for 12–14 cycles. The enriched libraries were purified using the solid-phase reversible immobilization method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). Sequencing on the Illumina GAII and HiSeq 2000 platform was performed at the Vincent J. Coates Genomic Sequencing Laboratory at the University of California, Berkeley, USA (UC Berkeley) to generate single-end 50, 76 and 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. BS-Seq: About 500 ng of genomic DNA was isolated from various honey bee developmental stages, fragmented by sonication, end repaired, and ligated to custom synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer's (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (QIAGEN), as outlined in the manufacturer's instructions. The bisulfite-converted libraries were then amplified by PCR using ExTaq DNA polymerase (Takara Bio) for 12–14 cycles. The enriched libraries were purified using the solid-phase reversible immobilization method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). Sequencing on the Illumina GAII and HiSeq platform was performed at the Vincent J. Coates Genomic Sequencing Laboratory at the University of California, Berkeley, USA (UC Berkeley) to generate single-end 76 and 100 base reads.