Extended Data Fig. 2: Nascent RNA sequencing (EU-Seq) computational analysis and controls, related to Fig. 1.
From: Genome-wide RNA polymerase stalling shapes the transcriptome during aging
a, EU-labelled nascent RNA sequencing (EU-seq) analyses flowchart. b, c, Sequence read distribution in EU-seq and total-RNA sequencing in introns and exons (b) and mapped on genes (c), showing increased number of intronic reads in EU-seq, indicating nascent RNA enrichment. d, Bioanalyzer plots of on-bead synthesized cDNA to generate EU-seq libraries. Since reverse transcriptase cannot synthesize cDNA through biotin covalently bound to DNA, cDNA is only generated between covalently EU-bound biotins or from EU-bound biotin to the RNA end. Adult and old cDNA is almost identical, indicating similar EU incorporation rates. e, table depicting EU incorporation for a range of distances between EU molecules as modelled by Poisson process. Three groups of RNA species were examined: 1) ≤300 nucleotides (n = 7932), 2) 1000 to 3000 nucleotides (n = 1983), and 3) 2000 to 4000 nucleotides (n = 1802). Shown is median and Interquartile Range (IQR). f, statistical and probabilistic framework table depicting 1.5-fold reduction for a range of EU incorporation distance differences. If EU incorporation differs between adult and old, it is expected that in RNA species ≤300 nucleotides the probability of at least one EU incorporation is significantly lower. For each specified pair of 1.5-fold apart intensities, the expected fold change in aged liver was calculated and the corresponding 7932-dimensional vectors of probabilities were compared by Mann-Whitney U-test. There is a very high, significant probability that a difference should be observed between adult and old (p < 2.2 × 10−16, column 3) if there is a 1.5-fold reduction in EU incorporation in aged liver. g, percentage sequence reads in EU-seq datasets mapping to RNA species length categories i) ≤300 nucleotides, ii) 1000 to 3000 nucleotides, and iii) 2000 to 4000 nucleotides. The ≤300 nucleotides length category shows a non-significant (p-value = 0.061093, two-sided unpaired t-test) 1.2-fold increase in aged liver, indicating that it is unlikely that EU availability is different between adult and aged livers. Data are mean ± SEM. h, percentage EU-labelled nascent RNA reads synthesized by different RNA polymerases (RNAPI-II-III, and mitochondrial RNAP (RNAP-MT)). Adjusted old values (orange) were calculated by proportional compensation of nascent transcription reduction as observed in Fig. 1a. n = 3 mice/group. Data are mean ± SEM. i, Alternative splicing in EU-seq data. n = 3 mice/group. Data are mean ± SEM. j, Ratios of splice donor and acceptor sites in EU-seq. n = 3 mice/group. Data are mean ± SEM.
