GSM3097276: K115_60min_cR40; Apis mellifera; RNA-Seq
Bee heads were separated from bodies on dry ice and placed in a dissection dish with pure ethanol and dry ice to prevent RNA degradation during dissection. The cuticle of the head capsule was removed and each head was placed in RNAlaterICE (Life Technologies) at -20°C for 14 to 18 h. The heads were then opened, the hypopharyngeal glands removed, and the whole brain removed. Using a fine scalpel, the optic lobes were removed and a horizontal incision was made across the midbrain through the posterior protocerebral lobe. The lower part of the midbrain containing the antennal lobes and the subesophageal ganglion were removed. The upper part of the midbrain containing mostly the mushroom bodies (MB) and the central brain was used for gene expression analysis. The MBs were placed in a new 1.5 mL tube and kept frozen (-80°C) until RNA extraction. RNA extraction was performed with a PicoPure RNA Isolation Kit (Applied Biosystems; Lot #: 1210063) and the standard protocol, including DNase treatment (QIAGEN) to remove genomic DNA contamination. 550 ng RNA from each sample (except for 19 samples that yielded 350- 550 ng) were used for whole mRNA expression analysis. RNA-seq libraries were constructed with the TruSeq® Stranded mRNA HT (high throughput kit, Illumina cat #: RS-122-2103) using an ePMotion 5075 robot (Eppendorf) The libraries were pooled in equimolar concentration as per instructions and each pool was quantitated by qPCR. Single-end sequencing (read length = 100nt) was performed on an Illumina HiSeq2000 using a TruSeq SBS sequencing kit version 3.
GEO Accession
GSM3097276