Targeted TimeLapse sequencing

MEF cells were grown at 37°C in DMEM containing 10% FBS and 1% P/S At approximately 60% confluence, the media was replaced with media supplemented with s⁴U (700 μM). After 2h, the cells were rinsed with PBS, resuspended in TRIzol reagent, and stored overnight at -80°C. Following chloroform extraction, total RNA was ethanol precipitated including 1 mM DTT to prevent oxidation of the s⁴U RNA, and washed with 75% ethanol. Total RNA was resuspended and treated with TURBO DNase, then extracted with acidic phenol:chloroform:isoamyl alcohol and ethanol precipitated and washed as described above. Isolated total RNA was added to a mixture of TFEA (600 mM), EDTA (1 mM) and sodium acetate (pH 5.2, 100 mM) in water. A solution of NaIO₄ (10 mM) was then added drop wise and the reaction mixture was incubated for 1h at 45°C. Potassium chloride (300 mM) and sodium acetate (pH 5.2, 300 mM) were added and the reaction mixture was allowed to stand on ice for 10 min. prior to centrifugation (>10000 rpm, 30 min, 4°C) to precipitate remaining periodate. The RNA in the supernatant was then ethanol precipitated and washed three times with 75% ethanol prior to resuspension in nuclease-free water. The chemically treated RNAs were then reverse transcribed using a mixture of mouse Actb and Gapdh-specific mRNA RT primers (see Supplementary table S1b). The resulting cDNA was then amplified with Phusion polymerase using corresponding forward PCR primers to produce PCR amplicons approximately 150 nt in length. An Illumina sequencing library was constructed using the Illumina TruSeq Index adapters. Paired-end 75 bp sequencing was performed on an Illumina HiSeq 2500 instrument. Sequencing reads were trimmed to remove adapter sequences and aligned to the mouse genome using Bowtie2²⁶. Aligned reads were parsed to identify mutations at each nucleotide position in the Actb and Gapdh mRNAs using a published software package.²⁷ Raw mutation probabilities were determined by dividing the number of recorded mutation events by the number of reads at that position. Mutation probabilities were normalized to appropriate control samples and filtered by read depth (only positions with depth > 3000 were included in analyses). Analyses and figure plot generation were performed in R using the tidyverse, corrplot, and multiplot packages^{28, 29}. The enrichment in mutation rates was tested for significance using a two-sided Wilcoxon test. Targeted sequencing was performed in duplicate using biologically distinct samples.

Targeted TimeLapse-seq of K562 RNA was performed similarly with the following exceptions. Cells were grown at 37°C in RPMI containing 10% FBS and 1% P/S. At approximately 50% confluence, the media was supplemented with a range of s⁴U concentrations (10 μM-40 μM) for 1h. Total RNA was isolated and chemically treated as described previously. The chemically treated RNAs were then reverse transcribed using a mixture of human MYC-specific mRNA RT primers (see Supplementary table S1b). A targeted sequencing library was prepared and analyzed as described above.

Further information concerning experimental design using biological materials can be found in the Life Sciences Reporting Summary.