RNA-seq of KD, rescues of NMD factors, and UPF1-flag CLIP-seq in HeLa cells.

This study aims at confidently identifying endogenous nonsense mediated decay (NMD) targets. To achieve this purpose, we performed KD of a few NMD factors in HeLa cells. Additionally, we performed rescue experiments for each factor, expressing an RNAi-resistant version of the gene from a plasmid. To determine transcripts bound by UPF1 in HeLa cells, A construct with a C-terminally flag tagged version of UPF1 was expressed. In order to avoid competition with endogenous UPF1, a KD was performed. Overall design: KD and rescue of three NMD factors (UPF1, SMG6, SMG7), plus a combined SMG6 and SMG7 KD, with individual rescues. 2 biological replicates of UPF1-flag CLIP-seq were carried out.