Illumina HiSeq 2500 paired end sequencing; Drosophila melanogaster selection lines confronted with Aspergillus nidulans
Drosophila melanogaster selection lines confronted with Aspergillus nidulans
--- Sampling took place at 24 hours post-confrontation start, where 52 larvae were pooled for one biological replicate collected from 4 independent dishes (balanced design with 13 larvae per dish). Only actively moving larvae were collected and sampling order was randomized. Larvae were snap frozen in liquid nitrogen.  Sample labels:  -sample number -selection regime and population number -treatment -replicate number (equals lane number)  --- Fly population: The Drosophila melanogaster fly stock used originates from a population established in 2006 from 113 isofemale lines. Flies were collected in the surrounding of Kiel, North Germany. Since then the population has been kept under moderate larval densities on a Drosophila breeding substrate composed of 62.5g yeast (Leiber GmbH Bramsche, Germany), sugar, and cornmeal, 12.5g Agar-Agar Kobe I, and 1L purified water. Eclosed flies of separate breeding bottles were brought together in one population box with several thousand flies and water and food supply.  --- Preparation sterile larvae: Flies were allowed to lay eggs on breeding substrate. Egg were washed down from the substrate and treated with 12% sodium-hypochlorite for 10 minutes, rinsed with autoclaved water and placed on sterile water-Agar, containing antibiotics (per liter Agar: 12.5g Agar-Agar Kobe I, 3g Nipagin 30mL 96% denaturated Ethanol). Petri dishes were equipped with an opening covered with 50µm gauze to enable ventilation and incubated over night at 25°C and darkness.   --- Fungal colonies/conidia: Aspergillus nidulans colonies were grown on malt-extract agar (30g malt extract, 5g peptone, 20g Agar-Agar Kobe I, filed to 1L with purified water; autoclaved) and incubated at 25°C for approx. a week. Conidia were then washed from colonies with saline-solution (8.6g NaCl, 300mg KCl and CaCl2, 1mL Tween80®, filled to 1L with purified water; autoclaved) and stored at 4°C. The A. nidulans strain RDIT2.3 and RJW 46.4 were used as wild type and delta-laeA mutant strain, respectively.  --- Fly population: The Drosophila melanogaster fly stock used originates from a population established in 2006 from 113 isofemale lines. Flies were collected in the surrounding of Kiel, North Germany. Since then the population has been kept under moderate larval densities on a Drosophila breeding substrate composed of 62.5g yeast (Leiber GmbH Bramsche, Germany), sugar, and cornmeal, 12.5g Agar-Agar Kobe I, and 1L purified water. Eclosed flies of separate breeding bottles were brought together in one population box with several thousand flies and water and food supply.  --- Preparation sterile larvae: Flies were allowed to lay eggs on breeding substrate. Egg were washed down from the substrate and treated with 12% sodium-hypochlorite for 10 minutes, rinsed with autoclaved water and placed on sterile water-Agar, containing antibiotics (per liter Agar: 12.5g Agar-Agar Kobe I, 3g Nipagin 30mL 96% denaturated Ethanol). Petri dishes were equipped with an opening covered with 50µm gauze to enable ventilation and incubated over night at 25°C and darkness.   --- Fungal colonies/conidia: Aspergillus nidulans colonies were grown on malt-extract agar (30g malt extract, 5g peptone, 20g Agar-Agar Kobe I, filed to 1L with purified water; autoclaved) and incubated at 25°C for approx. a week. Conidia were then washed from colonies with saline-solution (8.6g NaCl, 300mg KCl and CaCl2, 1mL Tween80®, filled to 1L with purified water; autoclaved) and stored at 4°C. The A. nidulans strain RDIT2.3 and RJW 46.4 were used as wild type and delta-laeA mutant strain, respectively.  --- 800 µL Trizol® were added and larvae tissue disrupted in BashingBead lysis tubes (2.0mm, ZYMO) for 2 x 30 sec. (30'000beads/min), chilled on ice between runs. Trizol® volume was filed up to 1.0mL and company instructions followed. 70% ethanol was added and samples loaded on RNA isolation columns (RNeasy mini Kit, QIAGEN). Manual instructions were followed. --- PolyA+ isolation, library preparation, and sequencing were performed by Eurofins Genomics (Ebersberg, Germany).
Experimental Factor: environmental history
breeding substrate inoculated with wild type A. nidulans
Experimental Factor: stimulus
none