(A) Left panels: Scatter plots showing the correlation between the number of crosslinked transcript copies (per million reads) recovered by PAR-CLIP and the expression level as measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The mean expression level for each transcript (obtained from biological duplicates) was normalized to the median expression level of the entire dataset, which consisted of all transcripts for which we recovered RNA-Seq reads (FPKM > 0) in a particular condition. Right panels: Histograms showing the gene expression distribution in RNA-Seq experiments. Deviations from the central tendency are color-coded, with warm colors closer to the central tendency and cool colors towards the tail ends of the distribution. The same color scheme is applied to the data points in the left panels. The dashed vertical lines indicate the standard deviation (SD) of the median-normalized data. (B) Waterfall plots showing the expression change in select transcripts identified by PAR-CLIP measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). Bars: mean values of biological duplicates. (C) Scatter plots showing the fold-change in the number of crosslinked transcript copies recovered by PAR-CLIP in the presence or absence of chemically induced ER stress and the fold-change in the number of RNA-Seq reads (FPKM) in the same conditions. Genes repressed or expressed during ER stress are indicated on the X-axis. Transcripts for which there is a loss or gain of recovered crosslinked copies are indicated on the Y-axis. (D) Representative quantitative real-time PCR measurements of the levels of select PAR-CLIP hit mRNAs in response to ER stress in different cell types. ER stress was induced with 300 nM of the ER calcium reuptake inhibitor thapsigargin, and the cells were pre-treated for 30 min with 5 μg/ml actinomycin D to block transcription. The IRE1 specific inhibitor 4μ8C (50 μM) was used as a control for specificity. Metrics for spliced XBP1 mRNA or the canonical RIDD substrate mRNA, BLOC1S1, were used as positive controls. Data: mean of triplicates. Error bars: SDs. (E) Changes in the transcript levels of select PAR-CLIP targets during ER stress measured by RNA-seq. Data: Mean FPKM values of biological duplicates. Error bars: 95% CI.