Fig. 2

Short labeling times proportionally enrich unstable transcripts. a DESeq2 [19] was used to identify features significantly enriched in 4tU-seq data from short labeling times (1.5 min and 5 min) compared to total RNA. The figure displays the percentage of transcripts in each category that was found to be significantly enriched (DESeq2 adjusted p < 0.05). For the DESeq analyses, all reads were considered. Thus for the intron-containing mRNAs we used reads that mapped to both introns and exons. b-d UCSC genome browser screen shots showing the change in distribution of reads at different labeling times (Y-axis), with annotation below in blue. SS indicates steady-state levels, generated by sequencing total RNA. b 4tU detects pre-rRNA precursors. Note that the total RNA sample is not shown because it was rRNA depleted. c 4tU-seq detects 3′ extended snR13 species. Data from an rrp6Δ strain are displayed for qualitative comparison. d Polycistronic precursor from which multiple snoRNAs are processed. Blue boxes represent the annotated mature snoRNAs. e Real time (RT) quantitative polymerase chain reaction (PCR) validation of the 4tU-seq results shown in (d). For the RT reaction, a reverse transcriptase primer was used that was complementary to the 3′ end of the snR72 snoRNA. This cDNA was then used to amplify the different amplicons shown below each bar plot (see the illustration in (d) for what each amplicon represents). The data were then normalized to the results obtained with rRNA-depleted total RNA (SS). 5'ETS 5' external transcribed spacer, 3' ETS 3' external transcribed spacer, 4tU 4-thiouracil, CUTs cryptic unstable transcripts, ITS internal transcribed spacer, ncRNA non-coding RNA, RP ribosomal protein, snRNA small nuclear RNA, snoRNA small nucleolar RNA, SUTs stable unannotated transcripts, tRNA transfer RNA