Cfx96 real time instrument

Co-IP of Isw1-Flag was performed as previously described (Gao et al., 2022 (link)). ISW1-FLAG was cultivated in YPD media overnight and subcultured to an A₆₀₀ of 0.8 (mid-log phase). 50 ml of the cell culture was added to 200 ml conical flasks containing 1% formaldehyde, and the flasks were incubated at room temperature with moderate rocking for 15 min. To end the cross-linking reaction, 2.5 M glycine was added to the mixture, which was then held for 5 min. At 4°C, cells were extracted at 1000 × g and washed twice with ice-cold PBS containing 125 mM glycine. After the chromatin is extracted, the DNA fragments are broken by ultrasound. The ultrasound condition is 3% power for 5 min (run for 3.5 s and then pause for 3 s). The fragment of the genome was then purified. IgG was used as the negative control for chromatin immunoprecipitation-qPCR. Quantitative real-time PCR (CFX96 real-time instrument; Bio-Rad) was used to analyze the gene abundance of immunoprecipitation using the specific primer pairs shown in Supplementary primers.

Total RNA was extracted using RNasy Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After determination of the purity and the integrity, total RNA, complementary DNA synthesis and quantitative real-time PCR reactions (RT-PCR) were carried out as previously described [19 (link)] using the CFX96 real-time instrument (Bio-Rad Laboratories Inc, Hercules, CA, USA). In hepatoma cells and HCC samples the relative expression was generated for each sample by calculating 2-Δ Ct [28 (link)]. Primers sequences used in the study are reported in Supplementary Table S2.

A 10-µL aliquot of prepared template was added to a master mix solution containing 5.0 µL of 4x Reliance One-Step Multiplex Supermix (Bio-Rad), 1.0 µL forward and reverse primers (5.0 µM), 1.0 µL 7.5 µM FAM-labeled Taqman Probe, and 2.0 µL nuclease-free water to a final volume of 20 µL (Supplementary Table S1 online) within a 96-well qPCR plate (Bio-Rad). RT-PCR standards, serving as positive controls, were included for each target and consisted of synthetic RNA covering the regions of interest in the SARS-CoV-2 genome. NP-tested negative clinical material was used as a host extraction and PCR control. Negative controls for the PCR reaction (NTC) were also included. Each prepared plate (1 per target) was sealed with optical tape then pulse centrifuged and placed in a CFX96 real-time instrument (Bio-Rad). After an initial reaction at 50°C for 10 minutes to complete reverse transcription, the samples were subjected to a cycle of 95°C for 10 minutes followed by 45 replicate cycles consisting of 2 steps programmed for 10 seconds at 95°C, then 30 seconds at 60°C. A plate-read step was included after each 60°C cycle. The limit of detection for this approach was 10 genomes per reaction or 1,000 genomes/mL starting material.