Bioruptor pico instrument

Total RNA was extracted from cells by using Trizol (Sigma). DNase
I-treated RNA (0.5–1 μg) was then reverse transcribed using
iScript cDNA Synthesis Kit (Bio-Rad). The sequences of gene-specific primers
used in qPCR are listed in Table 2.
Relative gene expression was calculated by using the
2^−ΔCt method where the expression of 18S gene was
used for normalization. For the RT-qPCR array, an RT² Profiler PCR
Array composed of 84 target genes was used (Qiagen, Human Type I Interferon
Response, PAHS-016Z) following the manufacturer’s recommended protocol.
Relative gene expression was first normalized to actin, which was included in
the array, then analyzed using the 2^−ΔΔCt method
by comparing ΔvIRF to WT KSHV-infected cells.
To measure the viral DNA load of KSHV-infected cells, the cells were
lysed in RIPA buffer, sonicated (1 cycle, 30 sec) using a Bioruptor Pico
instrument (Diagenode), and the total DNA was isolated by using
phenol-chloroform extraction. 10 ng of total DNA was used to measure the viral
copy number relative to host DNA. Viral DNA was amplified with KSHV
ORF11-specific primers while the host DNA was amplified using HS1-specific
primers (Toth et al., 2016 (link)) (Table 2). Analysis for PCR graphs was
compiled from at least three independent experiments.

Chromatin was prepared from hepatocytes for ChIP assays as previously described. Cells were fixed with 4% paraformaldehyde for 15 min, then glycine was added to a final concentration of 0.125 M and incubated for 10 min before harvesting. Chromatin was sonicated using a Bioruptor Pico instrument (Diagenode, Denville, NJ, USA). Chromatin preparations were subjected to ChIP using a ChIP-IT High Sensitivity Kit and Protein G Agarose Prepacked Columns (Active Motif, Carlsbad, CA, USA) using a PPARα (Abcam Ab24509) antibody. Normal rabbit IgG (Cell Signaling Technologies #2729S) and Histone H3 antibody (Cell Signaling Technologies #4620) were used as negative and positive controls, respectively. DNA was purified and concentrated using MinElute Reaction Cleanup columns (Qiagen). qRT-PCR and conventional PCR were performed using 2 μl of ChIP DNA samples from the 50 μl of purified samples using gene-specific primers (Table S1). Cycle threshold (Ct) values of ChIP and input samples were calculated and presented as fold change values.