title: small RNAs extracted from ovaries of 168 hours old flies (pooled from 50 individuals). Flies were housed for 120 hours at 18 degrees Celsius and then for 48 hours at 29 degrees Celsius..
design: A Drosophila melanogaster population (strain: Oregon RI, wildtype) was kept in culture tubes at 25°C for 48 hours. We then removed the adult flies and allowed the eggs to continue developing at 25°C. Adult female flies were collected 5 hours after hatching of the first adult fly to ensure same age and virginity of the collected individuals. The collected female flies were split into two groups (~150 individuals each) and transferred to new culture tubes that were kept at 18°C and 29°C respectively. Since the speed of development is subject to temperature, we sampled ovaries from 29°C flies after 48 hours and ovaries from 18°C flies after 120 hours to ensure that both groups had reached identical developmental stages. For each probe we pooled ovaries from 50 individuals. After the first sampling procedure, we switched the temperature for the remaining flies from 18°C to 29°C and from 29°C to 18°C, respectively. A second sampling procedure was performed after further 48 hours for flies now kept at 29°C and after 120 hours for flies now kept at 18°C; that is, flies in both populations were now 168 hours old.  Total RNA was extracted from pooled ovaries with TRI Reagent (Ambion) according to the manufacturer’s instructions. Together with GeneRuler Ultra Low Range DNA Ladder (Thermo Scientific) total RNA was applied to a 10% urea-based denaturing polyacrylamide gel and run for 20 minutes at 1300 V and 60 W. The sRNA fraction ranging from 15 to 35 nt was excised from the gel and resolved in 30 µl nuclease-free water using Ultrafree-MC and Ultra-0.5 3K centrifugal devices (Amicon) according to the manufacturer‘s instructions. For each probe, adapter molecules were ligated to both ends of the sRNA and the construct was subject to reverse transcription as previously described (Rosenkranz et al. 2015). We performed a PCR with individually tagged primers for each sRNA probe according to the following thermal cycling profile: 3' at 95°C, [30'' at 95°C, 30'' at 51°C, 20'' at 74°C] × 30, 5' at 74°C (forward primer for all probes: 5'-ACATGGACTGAAGGAGTAGA-3', reverse primer 18°C #1: 5’-ATTGATGGTGCCTACAG-3’, reverse primer 29°C #1: 5’-CTACGATTGATGGTGCCTACAG-3’, reverse primer 18°C #2: 5’-GACGGATTGATGGTGCCTACAG-3’, reverse primer 29°C #2: 5’-AGTAGATTGATGGTGCCTACAG-3’). We measured the DNA concentration of the obtained libraries with the Qubit 2.0 system (Invitrogen) and pooled the libraries by taking 30ng of DNA from each library. Sequencing of the pooled libraries was performed on an Illumina HiSeq 2000 system.