Faststart sybr master mix

Cellular total RNA was extracted and treated with DNase I (RNase-free, M0303S, NEB) to remove cellular DNA. For RT–qPCR of mt-mRNAs, 200–500 ng of RNAs was transcribed into cDNAs with PrimeScript RT Reagent Kit (Perfect Real Time, Takara, RR037A) and subjected to qPCR analysis with FastStart SYBR Master Mix (Roche) in a LightCycler 96 machine (Roche). Human ACTB and 18S rRNA were used as internal controls. The quantification of HepG2 cell mtDNA was conducted according to a published protocol^{41 (link)}. All primer sequences for RT–qPCR can be found in Supplementary Table 2.

To 18 μl of the fragmented RNA (with 2 μl Stop Buffer added) from the NAI-N₃-treated case (siALKBH7 versus siControl) above, 2 μl of 5 nM biotin-tagged ssDNA spike-in (Supplementary Table 2) was added and 2 μl was kept as input. Then the rest of the RNA solution was mixed with 20 μl of freshly prepared C1 bead suspension and the standard procedures described in the optimized icSHAPE protocol above were conducted. The collected RNA (serving as the pulldown sample, from the NAI-N₃-treated samples) and the input RNA were incubated with 1 μl 2 μM RT primer at 65 °C for 2 min and moved onto ice immediately. Then 4 μl 5× First Stranded Buffer (Thermo Fisher Scientific), 2 μl 10 mM dNTP Solution Mix (NEB), 1 μl 0.1 M DTT, 1 μl RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific) and 1 μl Superscript III Reverse Transcriptase (Thermo Fisher Scientific) were added to the RNA-primer mixture, followed by incubation of the reaction mixture (diluted into a 20-μl volume) at 42 °C for 5 min, 50 °C for 30 min, and 70 °C for 5 min. Then cDNAs were subjected to qPCR pipelines with FastStart SYBR Master Mix (Roche) in a LightCycler 96 machine (Roche) using appropriate primers for the flexible region and ssDNA spike-in (Supplementary Table 2).

HepG2 cells in 10-cm plates were prepared with cellular treatments such as siALKBH7 versus siControl. A 5-ml volume of culture medium with 0.5 mM EU was added to each plate and incubated at 37 °C for 10 min (ref. ^{43 (link)}; and also other time points such as 1–5 min or 10–60 min). Cells were washed once with cold DPBS and total RNA was immediately extracted with TRIzol and isopropanol precipitation, and then a biotin-tagged ssDNA spike-in was added. Cellular nascent RNA was enriched by biotin pulldown based on the standard protocol of the Click-iT Nascent RNA Capture Kit (Invitrogen, C10365). All RNA samples were denatured at 80 °C for 2 min before conducting RT with HIV Recombinant Reverse Transcriptase (Worthington). The cDNAs were subjected to qPCR pipelines with FastStart SYBR Master Mix (Roche) in a LightCycler 96 machine (Roche) using appropriate primers for junction regions and ssDNA spike-in (Supplementary Table 2).