Dntp solution mix

Reactions using purified DNA species as template were performed using Phusion™ polymerase along with dNTP solution mix and Phusion™ HF Buffer (all from New England Biolabs, Ipswich, MA). Reactions from cellular genomic DNA (colony PCR, multiplex allele-specific colony (MASC) PCR) were performed using 2x colony PCR master mix (Thermo Fisher Scientific, Inc., Waltham, MA).

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).