Supplementary Figure 8: SLAMseq robustly differentiates s4U-labeled and unlabeled transcripts in the context of total RNA.
From: Thiol-linked alkylation of RNA to assess expression dynamics
(a) Steady-state gene expression determined by Quant-seq generated from total RNA of mESCs subjected to s4U-metabolic labeling for 24 h in three independent cell cultures (r#1-r#3). All reads (sum of T>C containing and non-T>C containing) report steady-state gene expression. Spearman correlation coefficient (rS) and associated p-Values are indicated. (b) Relative abundance of background-subtracted T>C conversion-containing reads in Quant-seq libraries generated from total RNA of mESCs subjected to s4U-metabolic labeling for 24 h in three independent cell cultures (r#1-r#3). Spearman correlation coefficient (rS) and associated p-Values are indicated. (c) Relative abundance of labeled (T>C reads) and all reads (sum of T>C containing and non-T>C containing) shows uniform s4U labeling of transcripts in mESCs subjected to s4U-metabolic labeling for 24 h. Average of three independent cell cultures is shown. Spearman correlation coefficient (rS) and associated p-Values are indicated. (d) Treatment of unlabeled total RNA does not perturb gene expression values. Relative abundance of transcripts in Quant-seq libraries generated from total RNA of untreated mESCs before and after treatment with iodoacetamide. Average of three independent cell cultures is shown. Spearman correlation coefficient (rS) and associated p-Values are indicated. (e) Meta-analysis of three independent SLAMseq libraries generated from mESCs subjected to s4U-metabolic labeling for 24 h. Relative coverage across 250 nt counting windows at the 3′ end of transcripts, transcript U-content, as well as raw and U-content-normalized (norm.) conversion rates (Conv.) are shown for all counting windows expressed at steady-state >5 cpm (n=8408). (f) Conversion rates in counting window-mapping reads of Quant-seq libraries, prepared from mESCs before (no s4U) and after metabolic labeling for 24 h (+s4U) in three independent cell cultures are shown for all counting windows expressed at steady-state >5 cpm (n=8408). Dashed line represents expected background sequencing error rate. Outlieres are not shown. p-Value (Mann-Whitney test) is indicated.
