Quant ittm picogreen dsdna assay kit

Plant material for DNA extraction was grown under standard greenhouse conditions (16 h day/8 h night, 20°C). Young third leaves were sampled and immediately transferred into liquid nitrogen. Genomic DNA was extracted using guanidine thiocyanate-based DNA isolation in 96-well plate format according to Milner et al. (2018) (link). The DNA concentration of the samples was measured using Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. For accurate DNA quantification of higher number of samples, Quant-iT^TM PicoGreen^® dsDNA assay kit (Invitrogen, Carlsbad, CA, United States) and a Synergy HT microplate reader (BioTek, Bad Friedrichshall, Germany) were used.

DNA fragmentation was performed on a Covaris S2 Focused ultrasonicator with the following settings: duty cycle 10%, intensity 5, 200 cycles per burst during 190 s to obtain fragments with an average length of 200 bp. The power mode was set to frequency sweeping, temperature 6–8 °C and water level 12. A maximum of 3 μg DNA was dissolved in 130 μl TE and loaded in a microtube with AFA intensifier (Covaris). DNA was then analyzed on the Agilent 2100 Bioanalyzer (Agilent Technologies) and fragment distribution was analyzed on a high sensitivity DNA chip. Methylated DNA was captured using the MethylCap kit (Diagenode). The concentrations of the fragmented and captured DNA was determined on a Fluostar Optima plate reader (BMG Labtech) with the Quant-iTTM Picogreen^® dsDNA assay kit (Invitrogen) at 480/520 nm. A second quality control was performed after fragmentation on an Agilent 2100 HS DNA chip.

Review of all H&E slides was performed by a pathologist to confirm the tumor type, and to ensure that the sample was representative of the tumor and that normal surrounding tissues were not included. Genomic DNA was extracted from 10–20 6-µm-thick slides per FFPE block, depending on the tumor size. Purification of genomic DNA was performed using the QIAamp DNA FFPE tissue kit (#56404; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. De-paraffinization with xylene and ethanol was carried out as follows. After incubation for 5 min with 1 ml xylene, the samples were centrifuged for 1 min at 12,000 rpm, and the supernatant was removed without disturbing the pellet. The samples were then washed with 1 ml of absolute ethanol in order to remove the remaining xylene, and the pellet was air-dried for 10 min to allow the residual ethanol to evaporate. The pellet was lysed by incubation with 0.2 mg of proteinase K overnight at 60°C then subjected to column purification. Each genomic DNA sample was eluted in 50 µl of DNase- and RNase-free water, quantified using the Quant-iTTM PicoGreen dsDNA Assay kit (Invitrogen/Life Technologies, Grand Island, NY), and normalized to a 5 ng/µl concentration.