Quantifying the transcriptional effects of lncRNA depletion in HeLa cells with RNA-seq

Long noncoding RNAs (lncRNAs) are a major transcriptional output of the mammalian genome, yet their functions are poorly characterized. In this experiment, we used RNA interference (RNAi) and locked nucleic acid antisense oligonucleotides (LNAs) to deplete the lncRNAs LINC00899 and C1QTNF1-AS1 (C1) in HeLa cells. Both of these lncRNAs are of interest as their depletion results in mitotic delay, suggesting a role in mitotic progression. After depletion of each lncRNA with RNAi or LNAs, we performed high-throughput RNA sequencing and tested for differential expression compared to negative control samples (using control siRNAs or negative control LNAs) to identify downstream regulatory targets of each lncRNA. We generated 3-4 replicates for each condition, spread across multiple batches and experiments. Samples with the same batch number were treated at roughly the same time (within the same month), while the experiment number is nested within batch and refers to cells treated on the same day. Samples were pooled and sequenced across multiple lanes, yielding 8-9 technical replicates per sample.