FIGURE 1.
SLAM-seq workflow. Log phase cells are labeled with 4-tU followed by chasing the label with a high concentration of unmodified uracil. 4-tU is taken up by cells and incorporated into RNA. Samples are taken during the chase and metabolically inactivated using chilled ethanol. RNA is then extracted, and incorporated 4-tU is alkylated with iodoacetamide (IAA), yielding a stable derivative of the thiol-containing 4-tU. Reverse transcriptase misincorporates a guanine rather than adenine at derivatized positions during library construction, causing a T to C conversion. These are then quantitated using the SLAMDUNK pipeline.
