Easy characterization of transcription by Chromatin-Associated Transcript sequencing

In the last decade, new high-throughput sequencing techniques have revealed the complexity of the human transcriptome, allowing the characterization of long non-coding (lnc)RNAs. Since their expression has been reported as very specific to tissue, developmental stage and pathological variations, some lncRNAs have been proposed as biomarkers for diagnosis as well as prognosis of tumors. In this project, we investigate a role for the poorly characterized class of lncRNAs, antisense lncRNAs, and their association to the epithelial-to-mesenchymal transition (EMT), a biological process that promotes metastasis. In order to measure the transcription and properly describe the gene-structure of unannotated lncRNAs at the genome-wide level, we adapted a protocol of subcellular fractionation from Gagnon et al. (2014) to isolate nascent chromatin-associated transcript. This was performed on an in vitro model (Castro Vega et al, 2015) in which primary HEK cells naturally undergo EMT. Cells were immortalized prior and after EMT, therefore giving rise to the Epi (HA5-Early) and Mes (HA5-Late) cell-lines, which respectively display epithelial and mesenchymal phenotypes. In addition to the chromatin fraction, the cytoplasmic fraction of the cells was also sequenced in order to decipher the complexity of the transcriptome and assess the composition of mature transcripts. Overall design: The experiment was performed in biological duplicates on each cell-line (Epi and Mes) and RNA from both cytoplasmic and chromatin fraction was sequenced.