title: Sample 12
organism: Drosophila melanogaster
description: Protocols: --- 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-09T04:03:46Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-04-29T21:24:29Z
attributes.tag: External Id
attributes.value: SAMEA6808418
attributes.tag: INSDC center name
attributes.value: University of Groningen Groningen Institute for Evolutionary Life Sciences
attributes.tag: INSDC first public
attributes.value: 2020-05-09T04:03:46Z
attributes.tag: INSDC last update
attributes.value: 2020-04-29T21:24:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9011:Sample 12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: larvae
attributes.tag: environmental history
attributes.value: breeding substrate inoculated with delta-laeA mutant A. nidulans
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-9011:Sample 12
attributes.tag: scientific_name
attributes.value: Drosophila melanogaster
attributes.tag: stimulus
attributes.value: wild type Aspergillus nidulans
attributes.tag: strain
attributes.value: L3; selection= sLA