Lysis buffer

All dissections were performed between 1–4 pm to limit gene expression differences due to circadian rhythm genes. To isolate the Malpighian tubules, the penultimate abdominal segment of a cold-anesthetized female was gently pulled with a micro-tweezer until the gut, Malpighian tubules, and ovaries were isolated from the carcass. Following removal of the ovaries and foregut, the Malpighian tubules (and some hindgut) were then immediately transferred into lysis buffer (Zymo Research, Irvine, USA) and homogenized with a micropestle (Additional file 1: Table S1). Total RNA (and DNA in parallel) was then extracted from individual females or pools of 8–12 Malpighian tubules per biological replicate using the ZR-Duet kit (Zymo Research) with an on-column DNAse treatment step.

Fecal samples of the KETO study were processed using a previously described combination of enzymatic digestion and mechanical disruption by bead beating.⁶⁴ (link) Briefly, 300μL of the fecal slurry were centrifuged and the pellet dissolved in 800 μL enzyme mix A (5 μL of Lysozyme 10 mg/mL, 13 μL Mutanolysin 11.7 U/μL, 3.2 μL Lysostaphin 1 mg/mL, 778.8 μL 1x PBS) and transferred to a MP lysing matrix B tube (0.1 mm silica spheres, MP Biomedicals). The enzymatic digestion was initiated by incubation at 37°C for 30 minutes. A second enzymatic step was performed by adding 62 μL of enzyme mix B (10 μL Proteinkinase K 20 mg/mL, 50 μL SDS 10%, 2 μL RNase A 10 mg/mL) and incubation at 55°C for 45 minutes. Mechanical lysis was performed by bead beating at 6m/s for 40 seconds (FastPrep-24, MP Biomedicals, Solon).
For the CARBFUNC study, 100-150 mg of the fecal samples were mechanically lysed for 40 seconds at 6m/s in MP lysing matrix B tubes (0.1 mm silica spheres, MP Biomedicals) containing 700μL lysis buffer (Zymo Research). Metagenomic DNA was isolated from lysates using the ZR Fecal DNA Miniprep Kit (Zymo Research) according to the manufacturer’s recommendation. The DNA was eluted in 100 μL RNase-free water and stored at -20°C.

Nematodes were supplemented with EPA and infected with C.albicans as described above. Following this, nematodes were washed by centrifugation at 4000g for 2 min, the supernatant gently aspirated and 2 ml RNAlater (Invitrogen) added to each sample to combat degradation of RNA. Samples were frozen at − 80 °C until RNA extraction. Samples were thawed on ice, centrifuged at 4000g for 2 min to collect cells and RNAlater was aspirated. The nematode pellet was resuspended in 600 µl of lysis buffer (Zymo Research), supplemented with 1 volume of glass beads (diameter 0.5 mm) and mechanically homogenised twice for 15 min using a Disruptor Genie Analog Cell Disruptor. Total RNA was extracted from samples using Quick-RNA MiniPrep kit (Zymo Research), including removal of the genomic DNA by DNase digestion, according to manufacturer’s instructions. The RNA samples were evaluated using the Thermo Scientific NanoDrop ND-1000 Ultraviolet Visible Spectrophotometer to determine total RNA concentration in each sample.

Alveolar macrophages or BMDMs were lysed in lysis buffer (Zymo Research) followed by RNA isolation using the Quick-RNA MicroPrep Kit (Zymo Research) according to the manufacturer’s instructions. Isolated RNA was reverse transcribed using a cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Realtime/qPCR was performed using SYBR Green Master Mix (Roche) according to the manufacturer’s instructions. Transcripts were normalized to two housekeeping genes (GAPDH and ß-actin). The relative expression was calculated with the 2^−ΔCT method. Primer sequences are listed in Supplementary Table 1.

RNA extraction and cDNA synthesis of samples stored in DNA/RNA shield were performed as described previously (9 (link)). In brief, samples were thawed at RT and resuspended in an equal volume of lysis buffer (Zymo Research) before starting the Quick-DNA/RNA miniprep (Zymo Research) protocol following the manufacturer’s instructions. After a TURBO™ DNase (Thermo Fisher Scientific) treatment step, purity of RNA samples was checked using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific) and cDNA synthesis was carried out using the SuperScript™ IV First-Strand Synthesis System (Invitrogen™) with oligo(dT)₂₀ primers (Invitrogen™).