Quant it picogreen dna assay

A set of primers adapted to massive sequencing for the Illumina technology was used to amplify the 16S rRNA gene (V3-V4 hypervariable region^{46 (link)}) from the metagenomic DNA in a PCR reaction. PCR reactions were performed with 30 ng of metagenomic DNA, 200 μM of each of the four deoxynucleoside triphosphates, 400 nM of each primer, 2.5 U of FastStart HiFi Polymerase, and the appropriate buffer with MgCl₂ supplied by the manufacturer (Roche, Mannheim, Germany), 4% of 20 g/mL BSA (Sigma, Dorset, United Kingdom), and 0.5 M Betaine (Sigma). Thermal cycling consisted of initial denaturation at 94 °C for 2 minutes followed by 35 cycles of denaturation at 94 °C for 20 seconds, annealing at 50 °C for 30 seconds, and extension at 72 °C for 5 minutes. Amplicons were combined in a single tube in equimolar concentrations. The pooled amplicon mixture was purified twice (AMPure XP kit, Agencourt, Takeley, United Kingdom) and the cleaned pool requantified using the PicoGreen assay (Quant-iT, PicoGreen DNA assay, Invitrogen). Subsequently, a sequencing library was prepared under a pair-end configuration following manufacturer’s instructions, and sequencing on the Illumina MiSeq platform was performed at Life Sequencing SL facilities (Valencia, Spain). Sequencing statistics and rarefraction curves are shown in Supplementary Table S1 and Supplementary Figure S2, respectively.

DNA content was determined using the Quant-iT PicoGreen DNA assay (Invitrogen, Life Technologies, United Kingdom). Briefly, 10 μL of cell lysate (in 0.1% Triton™ X-100) was added to 90 μL of TE (10 mM Tris-HCl, 1 mM EDTA) buffer. 100 μL of PicoGreen reagent was added to all samples for 5 min. The fluorescence was then measured in a SPARK spectrophotometer (TECAN, CH) at 480/520 nm wavelength.

Cells were cultured in 96-well plates (3 × 10³ cells/cm²) in a basal medium for 24 h. The medium was replaced with a fresh basal medium supplemented with/without AZT (10 μM), DFO (10 μM) or AZT (10 μM)/DFO (10 μM) for 24 h, followed by 3 days of basal culture. The detection of H3K9 acetylation and methylation was performed using the EpiQuik^TM In Situ Histone H3-K9 Acetylation Assay Kit (Epigentek, New York, NY, USA) and EpiQuik^TM In Situ Histone H3-K9 Methylation Assay Kit (Epigentek, New York, NY, USA) according to the manufacturer’s protocol. The absorbance was read in a SPARK spectrophotometer at 450 nm. Histone acetylation and methylation was normalised with the DNA content.
The DNA quantification was determined by a Quant-iT PicoGreen DNA assay (Invitrogen, Life Technologies, Paisley, UK). Briefly, cells were lysed following three freeze-thaw cycles in 0.1% Triton^TM X-100 in phosphate-buffered saline (PBS, Lonza, Manchester, UK). A buffer of 90 μL of TE (10 mM Tris-HCl, 1 mM EDTA) was added to 10 μL of cell lysate in a 96-well plate (Corning, Deeside, UK). All samples received 100 μL of PicoGreen reagent and were incubated for 5 min. Subsequently, fluorescence was measured in a SPARK spectrophotometer at an excitation/emission wavelength of 480/520 nm.

A set of primers adapted to massive sequencing for the Ion Torrent platform (Lifetechnologies) were used to capture 16S (modified from a previous study42 (link)) and 18S (modified from a previous study43 (link)) rRNA from the solar-panel DNA extraction in a PCR reaction. PCR reactions were performed with 30 ng of metagenomic DNA, 200 μM of each of the four deoxynucleoside triphosphates, 400 nM of each primer, 2.5 U of FastStart HiFi Polymerase, and the appropriate buffer with MgCl2 supplied by the manufacturer (Roche, Mannheim, Germany), 4% of 20 g/mL BSA (Sigma, Dorset, United Kingdom), and 0.5 M Betaine (Sigma). Thermal cycling consisted of initial denaturation at 94 °C for 2 minutes followed by 35 cycles of denaturation at 94 °C for 20 seconds, annealing at 50 °C for 30 seconds, and extension at 72 °C for 5 minutes.
Amplicons were combined in a single tube in equimolar concentrations. The pooled amplicon mixture was purified twice (AMPure XP kit, Agencourt, Takeley, United Kingdom) and the cleaned pool requantified using the PicoGreen assay (Quant-iT, PicoGreen DNA assay, Invitrogen). Subsequently, sequencing on the Ion Torrent platform was performed at LifeSequencing S.L. (Valencia, Spain).