1.4 mm ceramic beads

Fly surfaces were sterilized by washing twice in 95% EtOH followed by washing twice in sterile water. Single flies were placed in tubes with 1.4 mm ceramic beads (Qiagen; MD, USA) and ATL buffer from PowerMag Bead Solution (Qiagen) and homogenized with a bead mill homogenizer (Bead Ruptor Elite, Omni, Inc; GA, USA), following extended vortex for 45 min at 4 °C. Following an overnight proteinase K treatment (2 mg/mL) at 56 °C, DNA was extracted using the MagAttract PowerSoil DNA EP Kit (Qiagen) according to the manufacturer’s instructions. 16S rRNA gene of bacteria was identified by PCR amplification with primers to the V3-V4 region (515F: GTGYCAGCMGCCGCGGTAA; 806R: GGACTACNVGGGTWTCTAAT)^{87 (link)}. The primers contain a 12-base pair Golay-indexed code for demultiplexing. PCR reactions were performed with the KAPA3G Plant kit (Sigma Aldrich, MO, USA) under the following conditions: 95 °C for 3 min, followed by 35 cycles of 95 °C for 20 s, 50 °C for 15 s, 72 °C for 30 s, and a final extension for 72 °C for 3 min. The PCR products were purified and normalized using the Just-a-plate kit (Charm Biotech, MO, USA). High throughput sequencing (HTS) was performed with Illumina MiSeq and 250 bp paired-end kits (Illumina, Inc., CA, USA).

C. elegans (N2) gravid adults (~15–20) were bleached onto 60mm RNAi plates, eggs were allowed to hatch and grow to young adults at 15°C. For heat stress application, replicate plates were placed at either 15°C or 37°C for 2 hr. At the end of the heat stress, worms were collected in S-Basal, and pellets were frozen at –80°C. Four independent biological replicates were collected. To prepare the samples for LC-MS, the pellet was thawed on ice and washed with 0.9% NaCl. Washed worms were then transferred to 2 mL FASTPREP tubes (MP Biomedicals) containing 1.4 mm ceramic beads (Qiagen). The samples were then resuspended in 1 mL 80% methanol (LC-MS grade) and homogenized using a bead beater (6.5 m/s; 20 s). The samples were cooled on ice between cycles. The homogenized samples were then vortexed at 4 °C for 10 min and centrifuged at 21,000 RPM for 10 min at 4 °C. The supernatant was removed at dried under vacuum. The pellet was resuspended in ice cold RIPA buffer and vortexed at 4 °C for 10 min and centrifuged at 21,000 RPM at 4 °C for 10 min. The supernatant was removed and used for protein quantification using Pierce Protein BCA assay kit (ThermoFisher). The protein quantification was then used to resuspend the pellet for an equal input of 0.5 μg/ml of protein per sample.

Lumbosacral DRG neurons (L5-S1) were homogenized using a MagNa Lyser Instrument (Roche Diagnostics, Mannheim, Germany) and 1.4 mm ceramic beads (Qiagen, Hilden, Germany)). Total RNA was extracted from L5-S1 DRG tissue using the RNAqueous^®-Micro kit (Applied Biosystems, Bedford, MA, USA). An aliquot of each RNA sample was run on a denaturing agarose gel to assess integrity. Intact RNA was then quantified by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), followed by treatment with DNase. DNase-treated RNA (500 ng) was reverse transcribed using the NZY M-MuLV First-Strand cDNA Synthesis Kit (NZYtech, Lisbon, Portugal), according to the manufacturer’s instructions. cDNA was diluted and stored at −20 °C for later use.