Quant it picogreen double stranded dna dsdna assay kit

A Quant-iT™ PicoGreen™ double-stranded DNA (dsDNA) Assay Kit (Fisher Scientific, Loughborough, UK) was used to estimate the cell number. After washing samples with PBS, scaffolds were placed in eppendorf tubes containing 1% Triton X-100 (Sigma Aldrich, Gillingham, UK) in distilled water. Control cells were adherent to the well plate and were removed by scraping before being added to Eppendorf tubes. Thereafter, the cells were rapidly freeze–thawed 3 times using −80 °C freezers. After the final thaw, 100 μL from each sample was added to a 96-well plate in triplicate. A standard curve was also generated using a lambda DNA standard (Thermo Fisher Scientific, Loughborough, UK). A total of 100 μL of PicoGreen solution (0.005% Picogreen concentrate (Thermo Fisher Scientific, Loughborough, UK) was diluted in a 5% tissue buffer (Fisher Scientific, Loughborough, UK) in dH₂O) and added to each sample as well as the standard curve. Ninety-six well plates were kept in the dark and left to react for 5 min until they were read using an M200 plate reader (Tecan UK, Reading, UK), and V7.2 Magellan analysis software was used with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Complete nucleic acids were extracted from soils using a modified cetyltrimethylammonium bromide (CTAB) procedure, as per DeAngelis et al. (16 (link)). This method has two bead-beating steps, which are expected to reduce extraction bias against fungi (26 (link)). The three extractions were then pooled, and DNA and RNA were separated using an AllPrep DNA/RNA kit (Qiagen). DNA was quantified using the Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Invitrogen), according to the product instructions.
Shotgun metagenome library preparation, sequencing, assembly, and annotation were completed at the Joint Genome Institute using standard operating procedures and pipelines. Unamplified libraries were prepared for each sample using a modified version of the Illumina TruSeq protocol with 500 ng of purified soil DNA. DNA was mechanically sheared to a median length of 270 bp using a Covaris LE220 and then size-selected using solid-phase reversible immobilization. Fragments were then end repaired, and poly(A) tails were added prior to ligation to barcoded sequencing adaptors. After the quantification of individual libraries using quantitative PCR, 11 to 12 libraries were pooled for sequencing on an Illumina HiSeq 2000 (2 × 150-bp strategy). This resulted in an average ± standard error of the mean (SEM) of 1.387 ± 0.06333 Gb per soil sample (metagenome).