title: 60_ST_t24_r4
organism: Drosophila melanogaster
description: Protocols: --- Sampling took place at 3, 6, 12, and 24 hour post-confrontation start, where 50 larvae were pooled for one biological replicate collected from 5 independent dishes (balanced design with 10 larvae per dish). Only actively moving larvae were collected and sampling order was randomized. Larvae were snap frozen in liquid nitrogen. --- 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 culture medium (DCM) 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 and 1g Chloramphenicol in 20mL 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 mutant strain respectively. --- Drosophila culture medium (DCM) composed of 62.5g yeast (Leiber GmbH Bramsche, Germany), sugar, and cornmeal, 12.5g Agar-Agar Kobe I, and 1L purified water was boiled, autoclaved at 121°C for 25 minutes, and 4 mL filled into Petri dishes (35 mm diameter) under sterile conditions. Petri dishes for control- (CO), wild type- (WT) and mutant-strain-confrontations (LA) were pre-treated with 50µL Acetone, which were allowed to evaporate for approx. 15 minutes. Fungal colonies were inoculated by placing a 1µL droplet of saline-solution in the middle of the substrate, containing 1000 conidia of the wild type or ∆laeA-mutant (titer adjusted using haemocytometer Neubauer improved 0.1mm depth). Units for mycotoxin confrontations (ST) were treated with 50µL Acetone containing 2µg sterigmatocystin (Sigma Aldrich) each. After approx. 15 minutes for evaporation 1µL sterile saline-solution was placed on the substrate. Units were incubated at 25°C and darkness for 3 days. Then, 15 sterile first instar larvae (see growth protocol) were transferred to each confrontation unit with autoclaved fine brushes. To prevent larvae from leaving/moving between dishes yet enable ventilation, dish opening was covered with a layer of autoclaved paper towel and closed with Petri dish lid. --- 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. --- totalRNA sequencing library preparation protocol: --- mRNA purification: Vortex beads vigorously in each step to resuspend beads and wash contaminates off Note: you may also start with 100ng mRNA, and directly go to section 2. During this purification the mRNA get extracted twice from the same beads. 1) Dilute 1-10μg total RNA with nuclease-free H2O to 50 μL in a 1.5 mL RNase free non-sticky tube. 2) Heat at 65°C for 5 minutes to disrupt the secondary structures, and place on ice. 3) Meanwhile, vortex well and aliquot 15 μL of oligo(dT) beads into a 1.5mL RNase free non-sticky tube. 4) Wash the beads twice with 100 μL Bead Binding Buffer. 5) Resuspend the beads in 50 μL Bead Binding Buffer, and add the 50 μL of totalRNA sample from step (2); rotate at RT for 5 minutes. 6) Remove the supernatant and wash the beads twice with 200 μL of Bead Washing Buffer. Vortex well to resuspend beads. 7) Add 50 μL of 10mM Tris-HCl to the beads, heat at 80°C for 2 minutes to elute mRNA from the beads. 8) Meanwhile aliquot 50 μL of Bead Binding Buffer to a fresh 1.5mL RNase free non-sticky tube. 9) After heating the beads and mRNA at 80°C for 2 minutes, immediately put on the magnet stand and transfer the supernatant (mRNA) to the binding buffer tube from step (8). 10) Heat the samples in the binding buffer at 65°C for 5 minutes to disrupt the secondary structures, and place on ice. 11) Mean while Wash the beads (same as from previous steps) twice with 200 μL of Bead Washing Buffer (Vortex Well). 12) Add 100 μL of mRNA sample from step (9) to beads and vortex to resuspend beads; rotate at RT for 5 minutes. 13) Remove the supernatant and wash the beads twice with 200 μL of Bead Washing Buffer. Vortex well to resuspend beads. 14) Add 17 μL of 10mM Tris-HCl to the beads, heat at 80°C for 2 minutes to elute mRNA from the beads. Immediately put on the magnet stand and transfer the supernatant (mRNA) to a fresh 200 μL thin wall PCR tube, and there should be ~8-16 μL of mRNA. --- RNA fragmentation Assemble the following mix on ice: 5x First Strand Buffer 4 μL, mRNA (50 ng or variable) x μL, Spiking RNA (optional) y μL, Nuclease free water (8-x-y) μL. Incubate at 94°C for 4 min and DIRECTLY put the tubes on ice. --- First strand synthesis In a PCR strip (0.2 mL) add: Fragmented RNA mix 12μL, Random Primers (3 μg/μL) 1μL, Incubate at 70 °C for 10min and snap cold on ice for 2 min. Add: DTT (100 mM) 2 μL, dNTP (10 mM each: A,C,G & T) 1 μL, RNaseOUT (40U/μL) 0.5 μL, H2O 2.5 μL = total volume 19 μL. Incubate the mix at 25 °C in a thermocycler for 2 min. Add 1 μL of Superscript III (200U/μL) and incubate with the following settings: 25°C for 10min, 50°C for 60 min, 70°C for 15 min, 4°C for hold. --- Second strand synthesis: Add: H2O 62.0 μL, 10x second strand buffer 10.0 μL, dNTPs (25 mM each: A,C,G & U) 4.0 μL, RnaseH (2U/μL) 1.6 μL DNA pol I (10U/ μL) 2.4 μL, total volume 100 μL, Incubate 2 hours at 16°C. Cleanup the sample with 180 μL XP beads and elute in 85 μL EB. --- Clean-up using XP beads: Place XP beads at ROOM temperature for at least 20 minutes before use! Add the appropriate amount of resuspended beads. Incubate at RT for 15 minutes (gently shaking/rotating). Put on magnet for 2 minutes. Remove the supernatant. Wash the beads twice with 200 μL 80% EtOH; Keep the tubes on the magnet, incubate each time for 30 secs. Remove the 80% EtOH and dry the beads for 15 minutes at RT. Resuspend the beads in the appropriate μL EB by pipetting. Incubate at RT for 2 minutes. Place the tube on the magnet for 2 min. Transfer the supernatant to a new tube --- End repair the dsDNA fragments: Add: dsDNA 85 μL, NEBNext End Repair Reaction Buffer (10X) 10 μL, NEBNext End Repair Enzyme Mix 5 μL total volume 100 μL, → Incubate at 20°C for 30 min. Cleanup with 160 μL XP beads and elute in 42 μL EB. --- A-tailing of the dsDNA fragments: Add: DNA sample from End repair 42 μL, NEBNext dA-Tailing Reaction Buffer (10X) 5 μL, Klenow Fragment (3' to 5' exo minus) 3 μL = total volume 50 μL. → Incubate at 37°C for 30 min. Cleanup with 90 μL XP beads and elute in 32.5 μL EB. ---- Adapter Ligation: Add: DNA sample from A-tailing 32.5 μL, Quick Ligation Reaction Buffer (5X) 10 μL, Adapter Oligo mix-with index 2.5 μL Quick T4 DNA Ligase 5.0 μL = total volume 50 μL. → Incubate at 20°C for 15 min. Clean-up the Ligated DNA with 50 μL XP beads and elute in 50 μL EB. Re-clean-up this with 50 μL XP beads and resuspend in 19 μL EB. --- User digest & Library pre-amplification: PCR components: Ligated DNA 19 μL, USER enzyme 1.0 μL, Primer cocktail 5.0 μL, 2x PCR master mix 25 μL = total volume 50 μL. PCR cyle: 37°C for 30 minutes (USER digest), 98°C for 30 secs, 15 cycles: 98°C for 10 secs, 60°C for 30 secs, 72°C for 30 secs, 72°C for 5 min, Hold at 4°C. Clean-up the PCR products with 50 μL XP beads and elute in 11 μL EB. Check the library on a High-sensitivity DNA chip.
attributes.tag: ENA first public
attributes.value: 2017-07-19
attributes.tag: ENA last update
attributes.value: 2016-12-13
attributes.tag: External Id
attributes.value: SAMEA26677168
attributes.tag: INSDC center alias
attributes.value: University of Muenster Institute for Evolution and Biodiversity (IEB) Animal Evolutionary Ecology Group
attributes.tag: INSDC center name
attributes.value: University of Muenster Institute for Evolution and Biodiversity (IEB) Animal Evolutionary Ecology Group
attributes.tag: INSDC first public
attributes.value: 2017-07-19T17:01:55Z
attributes.tag: INSDC last update
attributes.value: 2016-12-13T16:56:13Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5344:60_ST_t24_r4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: first instar larva stage
attributes.tag: growth condition
attributes.value: Sterigmatocystin
attributes.tag: sample name
attributes.value: E-MTAB-5344:60_ST_t24_r4