GSM3611059: LTS-IR03_298bp: MAm; Solenopsis invicta; RNA-Seq
We used a droplet of honey to attract workers in the forage area and collected workers of ca. 1.5 cm in length. Workers were decapitated one-by-one on a pre-chilled -20oC metal block sitting on ice. Worker heads were placed individually in 5 ul cold RNAlater ICE (AM7023, ThermoFisher) in a numbered 0.2 ml PCR-tube and temporarily stored in liquid nitrogen. Worker bodies were put in 2 ml tubes containing 50 ul QuickExtract solution (QE09050, Epicenter) and processed for Gp-9 genotyping. The processing of each individual, from sampling to head storage in liquid nitrogen was less than a minute. We sampled 96 individuals/polygyne and 50 individuals/monogyne colony each time. The head samples were stored in a -80oC freezer for subsequent antennae dissection. We sampled polygyne workers until we obtained at least 45 individuals of each genotype. We dissected the antennae of workers of the same genotype on a metal block sitting on ice, under a dissecting microscope (Leica, 20X magnification). We used a clean sheet of Kimwipe tissue to blot away any remaining RNAlater ICE on the samples before placing them into 200 ul cold TRIzol (15596018, Invitrogen). To minimize RNA degradation, we processed at most five heads at once. To minimize cross-sample contamination, we cleaned the metal block and forceps with RNase-Zap after each dissection. Antennae were stored in a -80oC freezer until RNA extraction. We extracted RNA using the Direct-zol RNA MiniPrep kit (R2050, ZymoResearch) following a modification of the manufacturer's instructions (Zhang et al. 2013). Frozen antennae in 200 ul Trizol were homogenized using a FastPrep-24 bead homogenizer (MP Biomedicals). Tissue destruction was checked under a microscope. If extra homogenization was required, we re-froze the sample in liquid nitrogen and repeated the homogenization (three times was the maximum needed). After the tissue was completely homogenized, we added an additional 400 ul Trizol into each sample, followed by vortexing for 10 secs and incubation at room temperature for 15 minutes. Samples were then phase separated with 120 ul chloroform. The aqueous phase was kept for RNA purification using the Direct-zol column with on-column DNase I treatment. RNA was eluted once in 25 ul RNase-free water. cDNA was amplified from each RNA samples using the Nugen Ovation V.2 kit (M01206, NuGEN Technologies), following the manufacturer's instructions. To prepare sequencing samples, we mixed four bioreplicates of amplified cDNA of the same genotype. Samples of the same bioreplicate contributed same amount of cDNA. follow the manufacturer's instructions of Illumina
GEO
GSM3611059
GEO Accession
GSM3611059