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. 2014 Nov 20;42(22):13615–13632. doi: 10.1093/nar/gku1186

Figure 5.

Figure 5.

4′Thiouridine-seq identifies genes repressed by dnTCF1EWT. (A) 4′Thiouridine-seq involves addition of 4′Thiouridine into cells for 30 min to label nascent RNA transcripts. 4′Thiouridine-labeled RNA is isolated and sequenced, allowing for snapshots of transcription at any given time point of dnTCF1EWT induction. (B) Comparison of AXIN2 levels as assessed by normal RT-PCR (before pulldown) versus 4′Thiouridine-RT-PCR (same sample after pulldown), n = 3. Pulldown of 4′Thiouridine-incorporated transcripts causes a more dramatic decrease in AXIN2 in response to dnTCF1E WT induction to be detected (n = 3). (C) Wnt target genes are greatly enriched after 4′Thiouridine pulldown when compared to the housekeeping gene UBA as assessed by RT-PCR. (D) The majority of genes that changed expression after 2 h of dnTCF1EWT induction (P < 0.02) and 9 h of dnTCF1EWT induction (P < 0.012) were downregulated. (E) The overlap of genes downregulated at 2 h post-induction and 9 h post-induction was highly significant (P-value denotes the result of a hypergeometric test). (F) The overlap of genes downregulated at 9 h post-induction in the 4′Thiouridine-seq and at 8 h post-induction in a previous microarray experiment in a different clonal DLD-1 cell line was highly significant (P-value denotes the result of a hypergeometric test).