Microcon 30

In-solution digestion of proteins was done as previously described^{86 (link)}. In brief, 20 μg of each sample were mixed with 75 μL 2% SDS, 1 mM Tris (2-carboxyethyl)phosphine and incubated at 55 °C for 1 h. Cysteines were blocked by adding 0.5 μL 200 mM methyl methanethiosulfonate and incubating 15 min at RT. After mixing with 200 μL 8 M urea in Tris pH 8.8, samples were transferred to Microcon-30 filter tubes (Millipore) and centrifuged at 14,000×g for 15 min at RT. Samples were washed four times with 200 μL 8 M urea and, subsequently, four times with 200 μL 50 mM ammonium bicarbonate by centrifugation as stated above. Proteins were digested with 0.7 μg Trypsin/Lys-C Mix (MS grade, Promega) in 50 mM ammonium bicarbonate overnight at 37 °C. Peptides were recovered by centrifugation. It was pooled with an additional wash with 200 μL 50 mM ammonium bicarbonate, dried in a speed vac and stored at −20 °C until used.

DNA was extracted from faecal samples using protocols based on the GuSCN⁄silica method [49 (link)] and further purified and concentrated through ultrafiltration using Microcon-30 (Millipore). Species identification was performed using previously developed species-specific primers [50 (link)]. All molecular analyses were undertaken in the Molecular Ecology Laboratory of the Doñana Biological Station (LEM-EBD).

DNA was co-extracted with the AllPrep DNA/RNA Mini kit (Qiagen) and eluted in 50–100 µL EB buffer. For some Low-Copy Number (LCN) samples, elution was additionally concentrated with Microcon-30 centrifugal filter devices (Millipore) to 20 µL.
DNA extracts were measured using the Quantifiler system (Applied Biosystems), with an ABI 7500 real-time PCR machine, according to the manufacturer's instructions.
Two microliters DNA (or up to 10 µL, if the 2 µL result was weak) were amplified with the Identifilerplus multiplex Kit (Applied Biosystems) in a total reaction volume of 25 µL on a GeneAmp PCR System 9700 (Applied Biosystems) according to the manufacturer's protocol.
PCR products were detected same as 2.8.

C. albicans yeast cells from the wild-type SC5314 and Δ/Δrlm1 mutant strain were inoculated into 10 ml of YPD and grown overnight at 30 °C. Each overnight culture was used to inoculate 20 ml of YPD to an initial OD640 of 0.4, and incubated at 30 °C for an additional 2 hours at 150 rpm. The cells were then harvested and immediately stored at −80 °C. RNA extraction was performed by using the hot acidic phenol method [23] . cDNA synthesis and labeling were carried out as described elsewhere [24] (link). Briefly, cDNA was synthesized from 40 µg of total RNA in the presence of 2-aminoallyl-dUTP. Samples were purified using Microcon-30 (Millipore) columns prior to coupling to NHS ester activated Cy3 and Cy5 fluorofores. Before hybridization, free dyes were removed using Chromaspin-30 (Clontech) columns and the efficiency of cDNA synthesis and dye incorporation was measured by spectrophotometry (NanoDrop). All samples had a degree of labeling (labeled nucleotides per 100 nucleotides) of around 5.0±1.5.

FASP was used with small modifications 17. In short, agarose beads from an IP experiment were gently vortexed in 75 μL 2% SDS, 1 μL 50 mM Tris (2‐carboxyethyl)phosphine at 55°C for 1 h. The reduced cysteines were blocked by incubation with 0.5 μL 200 mM methyl methanethiosulfonate for 15 min at RT. The sample was centrifuged at 16 000 × g for 20 min. The supernatant was mixed with 200 μL 8 M Urea in Tris pH 8.8, transferred to Microcon‐30 (Millipore, Lot R4NA17256), and centrifuged at 14 000 × g for 12 min at RT. The addition of 200 μL 8 M urea to the filter and centrifugation were repeated three times. To remove urea, four serial washes were performed with the addition of 200 μL 50 mM NH₄HCO₃ for each wash followed by centrifugation as stated above. Samples were digested with 0.7 μg Trypsin/Lys‐C Mix (MS grade from Promega) in 100 μL 50 mM NH₄HCO₃ overnight in a humidified chamber at 37°C. Hundred microliters of 0.1% acetic acid was added to the filter, and centrifuged. The tryptic peptides were collected, dried in a speedvac, and stored at −20°C until LC–MS analysis.