Gen5 data analysis software version 2

The NucleoSpin food kit (Macherey-Nagel, Düren, Germany), with some minor modifications according to the manufacturer’s instructions, was used to extract the DNA from the reference and validation model mixtures, as well as from commercial samples, with the addition of 2 μL of RNase (2 mg/mL) after the cell lysis step. The extractions were performed at least in duplicate assays using 200 mg of each sample. The extracts were kept at −20 °C until further analysis.
In order to evaluate the yield and purity of DNA extracts, UV spectrophotometric DNA quantification was performed on a SynergyHT multimode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA), using a Take 3 micro-volume plate accessory. The DNA content was assessed by the nucleic acid quantification protocol with sample type defined for double-strand DNA in the Gen5 data analysis software version 2.01 (BioTek Instruments, Inc., Winooski, VT, USA).

A UV spectrophotometer, using a Synergy HT multi-mode micro-plate reader (BioTek Instruments, Inc., Winooski, VT, USA) and the Take3 micro-volume plate accessory, was used to assess the yield and purity of DNA extracts. The nucleic acid quantification protocol for dsDNA samples in the Gen5 data analysis software version 2.01 (BioTek Instruments, Inc., Winooski, VT, USA) was used to determine the DNA content. The ratio of the absorbance at 260 and 280 nm (A260/A280) was determined as the purity parameter of the extracted DNA.
Electrophoresis in 1% agarose gel stained with 1× Gel Red (Biotium, Hayward, CA, USA) and ran in 1× SGTB buffer (GRISP, Porto, Portugal) for 20–25 min at 200 V was performed to evaluate the integrity of the DNA extracts. Agarose gels were visualized under a UV light tray Gel Doc™ EZ System (Bio-Rad Laboratories, Hercules, CA, USA) and a digital image was acquired with Image Lab software version 5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA).

The yield and purity of DNA extracts were assessed by UV spectrophotometry using a Take3 micro-volume plate accessory, on a Synergy HT multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The nucleic acid protocol was set for double-strand DNA in the Gen5 data analysis software version 2.01 (BioTek Instruments, Inc., Winooski, VT, USA), which was applied to absorbance data measured at 260 and 280 nm.
The quality of DNA extracts was further assessed by electrophoresis with 1% of agarose gel as previously described [22 (link)].

Prior to protein extraction, two buffers (PBS 0.2 M, pH 7.4 and Tris-HCl 100 mM, pH 8.0) were tested for their suitability to extract good quality protein from all model mixtures and commercial samples. Better protein extracts were obtained with Tris-HCl buffer (100 mM, pH 8.0), being the elected buffer for extracting proteins in this work. Briefly, 150 mg of sample were weighted and 1.5 mL of Tris-HCl buffer were added, followed by an incubation at 60 °C for 2 h, with stirring at 950 rpm and frequent vortexing to increase the protein yield. After incubation, the mixtures were centrifuged twice at room temperature (9000 g, 30 min). Between centrifugations, the supernatant was collected, and the pellet discarded in order to provide clear supernatants. After extraction, the protein concentration was assessed by UV spectrophotometry on a Synergy HT multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA), using a Take3 micro-volume plate accessory and the protein280 protocol in the Gen5 data analysis software version 2.01 (BioTek Instruments, Inc., Winooski, VT, USA).

The nucleosomes were quantified using the Cell Death Detection ELISAPlus kit (Roche Life Science, Indianpolis, IN, USA) according to the manufacturer’s instructions. This assay employs two murine antibodies directed at DNA (detection) and histones (capture). An eight-point standard curve was generated by twofold standard dilution of purified human nucleosomes (Human native nucleosomes; EMD Millipore, Billerica, MA, USA) in the incubation buffer from the enzyme-linked immunosorbent assay (ELISA) kit. Samples (18 uL) were assayed in duplicate. Standards and samples were applied followed by 80 uL of immunoreagent. Plates were incubated at (21 °C) for 2 h while shaking gently (300 rpm). Plates were decanted and washed thrice using 250 uL of incubation buffer/well. ABTS (100 uL) solution was added per well and incubated at room temperature with gentle shaking (250 rpm) for 15 min. The detection reaction was stopped with 100 uL of stop solution/well. The optical density of the wells at 405 nm were read on an Epoch microplate spectrophotometer (BioTek; Winooski, VT, USA). Standard curves were fitted to a five-parameter logistic curve using the GEN5 Data Analysis Software version 2.01 (BioTek) allowing interpolation for sample concentrations.