E tube

DNA was extracted from the Sterivex filters (i.e., 0.22- to 5-μm size fraction) using an AllPrep DNA/RNA minikit (catalog no. 80204; Qiagen) with a modified protocol: the filter paper removed from a Sterivex cartridge was put into a lysing matrix E tube (catalog no. 6914050; MP Biomedicals) with a mixture of 400 μL RLT plus buffer (containing 1% β-mercaptoethanol in accordance with the kit’s protocol) and 400 μL phenol-chloroform/isoamyl alcohol (25:24:1, vol/vol/vol); bead-beating was performed at 3,500 rpm for 30 s (MS-100; TOMY Digital Biology), followed by cooling on ice for 1 min, and then again at 3,500 rpm for 30 s; the supernatant after centrifugation (16,000 × g for 5 min at room temperature) was mixed with 500 μL chloroform-isoamyl alcohol (24:1, vol/vol) to remove the residual phenol and then centrifuged again; the supernatant was then used as the loading material for the AllPrep DNA spin column and processed in accordance with the manufacturer’s instructions. The quantity and quality of the DNA were measured using a Qubit double-stranded DNA (dsDNA) HS assay kit (catalog no. Q32851; Thermo Fisher Scientific) and a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific). Consequently, at least 2 μg purified DNA was obtained from each sample.

Nucleic acids were extracted based on the methods described in references 58 (link) and 28 (link) (Fig. 1a). Extraction buffer and phenol/chloroform (Ambion) (5:1 [pH 4.5]; 0.5 ml of each) were added to a lysing matrix E tube (M.P. Biomedicals) containing approximately 0.25 g of rumen fluid sample. Cells were mechanically lysed using a FastPrep machine (M.P. Biomedicals) (speed, 5.5; 30 s) followed by nucleic acid precipitation with polyethylene glycol (PEG) 8000. All steps were performed on ice or at 4°C. Nucleic acids were resuspended in 50 µl diethyl pyrocarbonate (DEPC) H₂O, and 1 µl of RNaseOUT (Thermo Fisher Scientific) was added. A 10-µl volume of nucleic acid extracts was subjected to DNase treatment (RQ1 DNase; Promega) and subsequent RNA purification (MEGAclear kit; Ambion). The quantity and quality of RNA were assessed via agarose gel electrophoresis and by the use of a NanoDrop spectrophotometer (ND-1000; Peqlab) and a Qubit assay kit (Thermo Fisher Scientific). The absence of DNA in the RNA preparations was verified by PCR assays targeting bacterial SSU rRNA genes and archaeal mcrA genes. The MessageAmp II bacterial kit (Ambion) was used according to the manufacturer’s manual to synthesize cDNA (via polyadenylation of template RNA and reverse transcription) and to perform in vitro transcription on the cDNA to amplify total RNA.

In order to facilitate the mold identification, colonies with different morphology were separated and sub-cultured on PDA plates until sporulation. The spore forming structure was examined under a microscope to identify the mold to the genus level, if possible, to narrow down the selection of species-specific primers for further identification [9 ,10 ]. Genomic DNA was extracted from the mycelia using a lysing matrix E tube (MP Biomedicals, Eschwege, Germany) and QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the protocols suggested by the manufacturer.
The extracted DNA samples that did not match with the selected primers were then multiplied using primers ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) and ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′), and subsequently sequenced on a Life Tech 3730xl DNA Analyzer (Thermo Fisher, Waltham, MA, USA) by the Beijing Genomics Institute, BGI Co., Ltd. (Shenzhen, China). The DNA sequence was then aligned with NCBI’s GenBank sequence database using a BLAST search with 99.99% of similarity, and the species identity was further validated using species-specific primers, if available in the US Environmental Protection Agency (USEPA) list of common indoor molds [9 ,10 ]. Primers used in this study are listed in Table 1.