Nucleospin bead tubes

Total DNA and RNA were co-extracted from 0.25 g soil (A_h horizon) following a modified protocol from Towe et al. [56 ]. Briefly, mechanical cell lysis using NucleoSpin Bead tubes (Macherey-Nagel, Germany) and the FastPrep-24 Instrument (MP Biomedicals, Germany; 40 s at 5.5 m s^-1) was combined with a phenol-chloroform extraction followed by a purification using an AllPrep DNA/RNA Mini Kit (Qiagen, Germany). The standard protocol was amended by an on-column DNA digestion step applying RNase-Free DNase Set (Qiagen). The high content of humic acids of the forest soil required an additional purification of the RNA by an RNeasy MinElute Cleanup Kit (Qiagen). RNA was checked for contaminating DNA by PCR amplification with primers 8f and Eub518 targeting the 16S rRNA gene [57 (link)] and subsequently transcribed with the GoScript Reverse Transcription System (Promega, Germany).

Preparation of cecal samples was performed on dry ice to avoid bacterial metabolism (including cutting for weighing). Around 10 to 20 mg of frozen cecal content (wet weight) was transferred into sterile tubes containing ceramic beads to allow cell disruption (NucleoSpin^® Bead Tubes, Macherey-Nagel, Dueren, Germany). One mL of pre-chilled methanol (−20 °C) was added and homogenised for 5 min with 30 Hertz using TissueLyser (Qiagen, Retsch, Germany). Then, the samples were centrifuged (10 min, 20 800 g, 4 °C) and the supernatants were transferred into sterile microcentrifuge tubes, placed on ice. Four-fold sample dilution in methanol and volumes of 50 µL were used for FT-ICR-MS infusion and UHPLC-MS/MS analysis, respectively. In addition, a pooled sample was generated from all samples (n = 26) and the storage was at −80 °C in tightly closed tubes.

Pooled L3 samples were washed five times with distilled water to remove ethanol. Genomic DNA was extracted using NucleoSpin^® Soil DNA extraction kit (Macherey-Nagel, Düren, Germany). Larvae were homogenised using NucleoSpin^® Bead Tubes with ceramic beads in the presence of SL1 lysis buffer (Macherey-Nagel) using a speedmill P12 (Analytik Jena, Jena, Germany). The lysates were transferred into new 1.5 ml Eppendorf tubes and DNA was extracted as instructed by the manufacturer. DNA was eluted in 50 µl elution buffer and stored at − 20 °C until used for further analysis.
Pooled adult males were treated as above and DNA was extracted using the NucleoSpin^® DNA Tissue extraction kit (Macherey-Nagel). Worms were homogenised using pestles in 1.5 ml microcentrifugation tubes containing 200 µl lysis buffer with proteinase K and incubated at 56 °C overnight with shaking (300× rpm). Then, lysates were transferred onto NucleoSpin^® columns followed by DNA purification as recommended by the manufacturer. DNA was eluted in 100 µl elution buffer and stored at − 20 °C.