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. 2019 May 28;8:e47040. doi: 10.7554/eLife.47040

Figure 1. Overview of PAR-CLIP experiments performed in this study.

(A) Overview of degradation pathways studied. (B) Number of high-confidence PAR-CLIP cross-link sites obtained for each factor after merging data from replicates.

Figure 1.

Figure 1—figure supplement 1. Biological replicate PAR-CLIP experiments have high correlation.

Figure 1—figure supplement 1.

(A) Total transcript occupancy of factors in replicate experiments are plotted (in log2 space) and Spearman correlation values are shown for each pair. Each dot corresponds to a transcript. The color indicates dot density. (B) Comparison of coverage profiles obtained from CRAC experiments of Xrn1, Mtr4, Trf4, and Ski2 in S. cerevisiae (Tuck and Tollervey, 2013) with occupancy profiles from our PAR-CLIP experiments highlights reproducibility of transcriptome profiles across different methods. These profiles show the averaged binding of degradation factors over mRNAs (sense strand: left and anti-sense strand: right) in a window of [±700 nt] around their transcription start site (TSS) and their poly-adenylation (pA) site in a window of [±700 nt]. Regions that have neighboring transcripts on the same strand were removed to avoid contaminating profiles (Materials and methods).
Figure 1—figure supplement 2. Western Blot analysis for all degradation factors analyzed in this study show IP efficiency.

Figure 1—figure supplement 2.

IP using the TAP-tag is detected by Western Blot analysis with tag specific antibody to show the IP quality of the different PAR-CLIP experiments representative for one replicate per factor. The factors are sorted according to their complexes: deadenylation (Ccr4, Pop2, Not1, Caf40, Pan3, and Pan3), decapping (Dcp1, Dcp2, Edc2, Edc3, and Dhh1), 5´→3´ exonuclease (Xrn1), exosome (Rrp6, Csl4, Rrp40, Rrp4, and Rrp44), TRAMP (Air1, Trf5, Mtr4, Air2, and Trf4), Ski (Ski2, Ski3, Ski7, and Ski8), and NMD (Upf1, Upf2, Upf3, and Nmd4). The molecular weight including the weight of the TAP tag (in kDa) is indicated for each factor. The band at ~25 kDa is caused by cross-reactivity of the light chain of the used antibodies for IP and Western Blot. A shift to higher molecular weight than indicated can be caused by UV-crosslinking of proteins to RNA.