Ensen and Roberts, 1948). For any first RNA-sequencing experiment we compared brain gene expression in queens that we artificially inseminated with either semen, seminal fluid or possibly a mock insemination treatment, exactly where no fluid was injected into the vaginal orifice. Virgin queens were sampled from their hives and randomly assigned to one of three experimental groups (known as `Experiment 1′): (i) instrumentally inseminated with six ml of semen pooled from approximately 10 males, (ii) instrumentally inseminated with 6 ml of seminal fluid, and (iii) SC-58125 Cancer mock-inseminated devoid of injecting any fluid. Inside a second RNA-sequencing experiment (referred to as `Experiment 2′) we further controlled for insemination of liquid in to the queen reproductive tract and assessed to what extent gene expression alterations in queen brains had been dependent on reception of semen or seminal fluid as opposed to on the mechanical stimulation of your reproductive tract upon insemination. To perform this, we randomly assigned queens to one particular of 3 experimental groups: (i) instrumentally inseminated with six ml of semen, (ii) instrumentally inseminated with six ml of seminal fluid, and (iii) instrumentally inseminated with six ml of Hayes saline (9 g NaCl, 0.two g CaCl2, 0.2 g KCl and 0.1 g NaHCO3 in 1000 ml H2O, adjusted to pH eight.7 and sterilised by filtration by means of a 0.22 mm syringe-filter, Membrane Solutions). To artificially inseminate queens, we sedated them with CO2 for any handful of seconds just before placing them within a holder mounted onto a regular artificial insemination instrument (Schley, Germany) and inseminating them based on the therapies. Queens have been afterwards allowed to recover for about 30 min ahead of we Dipivefrine hydrochloride Cancer returned them to their hives. We recollected queens immediately after 24 hr, narcotised them with CO2 for a few seconds and flash froze their heads in liquid nitrogen. All heads have been stored at 0 till dissections.Brain collection and RNA extractionBrains of queens had been dissected with Inox 5 watchmaker forceps under an Olympus SZX10 stereo microscope in ice-cold sterile Hayes saline. We ensured that brains were removed intact, which includes the optic lobes, and that all hypopharyngeal and other extraneous glandular tissue was removed. We combined the brains from three individual queens to get adequate RNA for Illumina TruSeq sequencing (see below), froze these pooled brain samples in liquid nitrogen and straight away stored them at 0 . We consequently obtained 3 biological replicates (each and every consisting of three pooled brains) per treatment group in both experiments 1 and 2 (18 samples in total; Supplementary file 15). To extract RNA from pooled brain samples, we briefly thawed them on ice, placed them in 10 ml of 0.25 M Tris pH 7.5 and homogenised them having a plastic pestle. We then added 50 ml of Trizol to each and every sample, incubated samples on ice for 15 min, completely vortexed and returned them to ice for a further 15 min, followed by centrifugation at 20,000 g for 15 min at 4 . Within the subsequent step, we collected the supernatant, added one volume isopropanol, briefly vortexed the samples and incubated them at area temperature for 20 min, followed by centrifugation at 20,000 g for 15 mins at 4 . Soon after discarding the supernatants, we washed each pellet with 70 EtOH,Liberti et al. eLife 2019;8:e45009..16 ofResearch articleEcology Evolutionary Biologyfollowed by air-drying and resuspension in 20 ml of RNase-free H2O. To precipitate the RNA, we added 1.six volumes of ice-cold four M LiCl and incubated sample.