Brutp

The NRO assay in HT29 cells was performed as described previously^{33 (link)} with minor modifications. In brief, HT29 cells were washed with cold PBS quickly and collected for NRO assay 24 h post nucleofection with ASOs. Collected cells were incubated in swelling buffer (10 mM Tris, pH 7.5, 2 mM MgCl₂, 3 mM CaCl₂) on ice for 5 min and were then collected by centrifugation. Cell pellets were subjected to lysis twice with 1.5 ml lysis buffer (10 mM Tris, pH 7.5, 2mM MgCl₂, 3 mM CaCl₂, 0.5% Igepal, 10% glycerol, and 2 U/ml RNasin Ribonuclease Inhibitor) to obtain purer nuclei. The resulting nuclear pellets were resuspended in 100 μl NRO buffer (50 mM Tris, pH 7.5, 5 mM MgCl₂, 150 mM KCl, 0.1% sarkosyl, 2 U/ml RNase inhibitor and 10 mM DTT) containing 0.1 mM ATP, GTP, CTP and BrUTP (Sigma). Transcription was performed for 3 min on ice and then 5 min at RT. The reaction was stopped by addition of 600 μl Trizol to extract RNA, followed by the DNase I (Ambion, DNA-free TM kit) treatment to remove genomic DNA. Purified RNAs were incubated with 2 μg anti-BrdU antibody (Sigma) or equal amount of IgG antibody (Sigma) at 4 °C for 2 h and were then immunoprecipitated with Dynabeads G pre-coated with yeast tRNA (Sigma). Precipitated RNAs were extracted by Trizol and were used for cDNA synthesis and qPCR analysis.

Each group of embryos (< 20) was washed twice in electrical permeabilization medium (EP: 0.25 M D-glucose, 100 µM CaCl₂.2H₂O, 100 µM MgSO₄, and 0.1%
polyvinylpyrrolidone) and finally in one change of transcription buffer (EP + 10 mM BrUTP; Sigma Aldrich). They were then transferred into a chamber between electrodes overlaid with a 20 µl
droplet of transcription buffer. Two 80 µsec electric pulses at 250 V/cm of direct current were triggered using an Electro Cell Fusion apparatus (Bex LF101L, Tokyo, Japan) with a 2 min
interval between pulses. Two minutes after permeabilization, embryos were cultured in mCZB medium for 1 h further under the same conditions as above. The embryos were then fixed in 4%
paraformaldehyde and new transcripts incorporating BrUTP were visualized by indirect immunofluorescence [15 (link)].

To locate the viral RNAs, a uridine analog, bromouridine 5′-triphosphate (BrUTP) (Sigma-Aldrich) that could be incorporated into RNA during its synthesis,^{52 ,53} (link) was used to label viral RNAs. In detail, GCRV-II-infected GCO cells at 15 h post infection (hpi) were treated with 10 μg/ml Actinomycin D (ActD, MCE) for 30 min and then transfected with BrUTP (Sigma) at a final concentration of 10 mM by using TransIntro® EL Transfection Reagent (TransGen Biotech) and then incubated in the presence of ActD for 1 h. The cells were fixed and permeabilized as described above and processed for indirect immunofluorescence.

Nuclei from hamster tissues were isolated as described in (32 (link)). Hamster BMDM and CHO nuclei were isolated using hypotonic lysis [10 mM Tris-HCl pH 7.5, 2 mM MgCl₂, 3 mM CaCl₂] with 0.1% and 0.5% IGEPAL, respectively. Nuclei were flash frozen and later 0.5–1 × 10⁶ nuclei in 200 μl GRO-freezing buffer [50 mM Tris-HCl pH 7.8, 5 mM MgCl₂, 40% Glycerol] were used in reactions with 3x NRO buffer [15 mM Tris-Cl pH 8.0, 7.5 mM MgCl₂, 1.5 mM DTT, 450 mM KCl, 0.3 U/μl of SUPERase In, 1.5% Sarkosyl, 366 μM ATP, GTP (Roche) and Br-UTP (Sigma Aldrich) and 1.2 μM CTP (Roche, to limit run-on length to ∼40 nt)] as described in (33 (link)). Run-on reactions were stopped, purified and GRO-seq and 5′GRO-seq libraries generated exactly as described in (31 (link)). BrU enrichment was performed using a BrdU Antibody (Sigma B8434-200 μl Mouse monoclonal BU-33) coupled to Protein G (Dynal 1004D) beads. For each sample, 3 × 20 μl of Protein G beads were washed twice in DPBS+0.05% Tween 20 (DPBS+T) and then the antibody coupled in a total volume of 1 ml DPBS+T under gentle rotation. About 1 μl of antibody was used per 8 μl of beads. Samples were amplified for 14 cycles, size selected for 160–250 bp and sequenced on an Illumina NextSeq 500 using 75 cycles single end.

NRO analyses were performed as described (Roberts et al. 2015 (link)). Nuclei (4 × 10⁶) from control or knockdown HEK293T cells were incubated for 30 min at 30°C in a buffer containing 10 mM Tris-HCl (pH 8.1); 2.5 mM MgCl₂; 150 mM KCl; 1 mM ATP, CTP, and GTP; 0.5 mM UTP; and 0.5 mM BrUTP (Sigma). BrU-labeled RNA was immunoprecipitated with 2 µg of BrdU antibody (BD Pharmingen, 555627) immobilized on protein G Dynabeads (Invitrogen). After treatment with DNase I (Sigma), the RNA was analyzed by RT-qPCR. Values of candidate genes were normalized to a BrU-labeled spike-in control RNA.

NRO was conducted as previously described (10 (link),11 (link),38 (link)) and used to measure the transcription activities of the MT genes upon MRE circRNA overexpression. In short, after subcellular fractionation, nascent transcripts in nuclei were labeled with bromouridine triphosphate (BrUTP, Sigma, B7166) for 5 min in NRO Buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 150 mM KCl, 0.1% (w/v) srkosyl, 10 mM dithiothreitol and 80 units/ml RNase inhibitor (Beyotime, R0102)), isolated by anti-BrUTP (Abcam, ab1893) following the standard immunoprecipitation procedure and subjected to RT-qPCR assays. Purity of nascent RNAs was confirmed by checking the relative pre-mRNA enrichment of MtnA and MtnB in NRO samples over that in whole-cell RNA extracts.