SLAMseq

Cultured cells are grown in 4-Thiouridine (S4U) for up to 24 h to allow S4U nucleotides to be incorporated into newly synthesized RNA until saturation is reached. Optimal concentration of S4U must be determined during exploring phase to avoid cell toxicity.

Cells are treated for >30 min with fast-acting compound or drug. The incubation duration depends on the compound and cell type. Negative controls need to be applied.

Cells are supplied with fresh media containing Uridine to perform a chase experiment to assess RNA decay over time.

Cells are sampled at a given timepoint(s) and RNA is extracted under reducing conditions. The isolated total RNA contains both existing (labeled) and newly synthesized (unlabeled) RNA for catabolic kinetics experiments.

Total RNA (1 - 5 µg) is mixed with iodoacetamide (IAA), which modifies the 4-thiol group of S4U-containing nucleotides via the addition of a carboxyamidomethyl group. The RNA is purified by ethanol precipitation prior to proceeding to library preparation.

Total RNA is reverse-transcribed to cDNA. For labeled RNA transcripts, reverse transcriptase incorporates a G instead of an A at positions where reduced *S4U-modified nucleotides are encountered.
Second-strand cDNA synthesis is then performed to generate a double-stranded SLAMseq RNA-Seq library.

PCR is used to amplify the SLAMseq RNA-Seq library and to add indices and full adapter sequences for next-generation sequencing. Sequencing reads with T>C mutations distinguish labeled from unlabeled transcripts, which are read-out during data analysis.