Fastdna spin kit for soil dna extraction

DNA was extracted from faecal samples and scraped colonic mucus using the Fast DNA™ SPIN kit for Soil DNA extraction (MP Biomedicals, USA) with the following modifications. The weight of faecal material was measured in tared tubes. The samples were resuspended in 978 μL of sodium phosphate buffer (provided) before being incubated at + 4 °C for 1 h following addition of 122 μL of lysis solution MT Buffer. The samples were then transferred into the lysing tubes and homogenised in a FastPrep® Instrument (MP Biomedicals) 3 times for 40 s at a 6.0 m/s speed with 5 min interval on ice between each bead-beating step. The protocol was then followed as recommended by the supplier.

Total microbial genomic DNA was extracted from 0.5 g of fresh soil using the FastDNA Spin Kit for Soil DNA Extraction (MP Biomedicals, USA). The quality of the extracted DNA was checked by 1% agarose gel electrophoresis, whereas the DNA concentration was determined using the NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, Wilmington, USA). All DNA samples were stored at −80°C. For each sample, the entire 16S rRNA gene was amplified by PCR using the primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′) and the following PCR program: 95°C for 3 min; 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 3 min; 72°C for 10 min; hold at 4°C. The PCR amplification of each sample was performed in triplicate to minimize the stochastic effect. The purified PCR products were pooled in equimolar amounts and then used to construct the amplicon library for the SMRT sequencing analysis using the PacBio sequencing platform (Novogene Co., Ltd., Beijing, China).

For preparation of DNA samples for microbiome sequencing, fecal samples were defrosted to room temperature and the FastDNA Spin Kit for Soil DNA Extraction (MP Biomedicals) was used for DNA extraction; 16S ribosomal RNA (rRNA) gene sequencing was undertaken at Glasgow Polyomics for analysis of the microbiome. In brief, primers that were specific to the V3 and V4 regions were used to generate 16S amplicons from 12.5 ng of extracted DNA using polymerase chain reaction. The resulting amplicons had dual barcodes and adapters added using the Nextera XT v2 adapter sets (Illumina). The libraries were combined in equimolar ratios and sequenced on a MiSeq (Illumina) instrument using a paired end, 2 × 300–base pair (bp) sequencing run. Samples were sequenced with an average of 50,000 reads.
For data analysis, FastQ files were generated from the sequencing data and quality-filtered using cutadapt (43 ). Cutadapt removes adapter sequences from high-throughput sequencing reads with a minimum quality of 25 and a minimum length of 250 bp. Paired-end reads were combined using PANDAseq and collated into a single FASTA file using the Qiime package (44 (link)). Further processing and analysis were completed using the Qiime wrapper and software.

The total genomic DNA was extracted using the FastDNA^® SPIN Kit for Soil DNA Extraction (MP Biomedicals, Santa Ana, CA) according to the manufacturer's instructions. The DNA was purified through Agencourt AMPureXPPCR Purification Beads (Beckman Coulter, USA). The integrity of genomic DNA was detected through agarose gel electrophoresis, and the concentration and purity of genomic DNA were detected via Qubit 3.0 Spectrophotometer. The V3-V4 hypervariable regions of the 16S rRNA gene and spike-ins were amplified with the primers 341F (5′-CCTACGGGNGGCWGCAG-3′)/805R (5′-GACTACHVGGGTATCTAATCC-3′) (Kataoka et al., 2019 (link)). The PCR amplification reaction was performed in triplicate in a total volume of 10μl. The reaction mixture consisted of 1 μl of 10 × Toptaq Buffer, 0.2 μl of Toptaq DNA Polymerase, 0.2 μl of each primer (10 μM), and 3 μl of template DNA. The following thermal cycling conditions were used: initial denaturation at 94°C for 2 min, 25 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The 16S rRNA gene amplicon was sequenced on the Illumina MiSeq platform at Genesky Biotechnologies, Inc. (Shanghai, China).

T. atroroseus strain IBT 11181 was obtained from the IBT Culture Collection at Department of Biotechnology and Biomedicine at Technical University of Denmark. It is also deposited in the CBS collection at CBS-KNAW, the Netherlands, as CBS 123796 and CBS 238.95. The T. atroroseus genome sequence has been deposited at DDBJ/ENA/GenBank under the accession LFMY00000000. The version described in this paper is version LFMY01000000. DNA sequences of T. atroroseus genes albA and talA are presented in S1 Appendix and S2 Appendix, respectively. Genomic DNA (gDNA) from T. atroroseus was extracted using the FastDNA^TM SPIN Kit for Soil DNA extraction (MP Biomedicals, USA), and T. atroroseus gDNA was used as PCR template for amplification of the up- and downstream fragments for deletion of talA (UA08_04451) and the green pigment gene (UA08_00425). T. atroroseus was cultivated in liquid- and on solid CYA medium (Czapek yeast autolysate) supplemented with 300 μg/ml hygromycin B (Hygrogold, Invivogen) when needed. Escherichia coli strain DH5α was used for plasmid propagation.

A list of all the strains used and produced in this study is provided in S1 Table. Aspergillus nidulans strain IBT 29539 (argB2, pyrG89, veA1, nkuAΔ)—referred to as NID1—was used for heterologous production of niduclavin and niduporthin. A. clavatus genomic DNA was obtained from strain IBT 12364 (NRRL 1) and was extracted using the FastDNA^TM SPIN Kit for Soil DNA extraction (MP Biomedicals, USA). Coding sequence of M. oryzae genes syn2 and rap2 were purchased from GenScript USA. Escherichia coli strain DH5α was used for plasmid propagation.
Aspergillus solid and liquid minimal medium (MM) and transformation medium (TM) was supplemented when necessary and according to strain genotypes with 4 mM L-arginine, 10 mM uridine, 10 mM uracil, and 1.3 mg/ml 5-fluoroorotic acid (5-FOA), and was prepared as described by Nødvig et al. [44 (link)]. E. coli DH5α was cultivated in Luria-Bertani (LB) medium consisting of 10 g/l tryptone (Bacto), 5 g/l yeast extract (Bacto), and 10 g/l NaCl (pH 7.0). LB medium was supplemented with 100 µg/ml ampicillin. All solvent used was of HPLC grade, and H₂O was purified and deionized by a Millipore system through a 0.22 µm membrane filter (MQ H₂O).