Identification and Functional Characterization of lncRNAs involved in Human Monocyte-to-Macrophage Differentiation

Long noncoding RNAs (lncRNAs) make up the largest portion of RNA produced from the human genome, but only a small fraction have any ascribed functions. Although the role of protein-coding genes in macrophage biology has been studied extensively, our understanding of the role played by lncRNAs in this context is still in its early stages. There are over 20,000 lncRNAs in the human genome therefore, attempting to select a lncRNA to characterize functionally can be a challenge. Here we describe two approaches to identify and functionally characterize lncRNAs involved in monocyte-to-macrophage differentiation. The first involves the use of RNA-seq to infer possible functions and the second involves a high throughput functional screen. We examine the advantages and disadvantages of these methodologies and the emergence of novel research and technologies aiding us in addressing these challenges. Overall design: For our candidate RNA-sequencing experiments we utilized our THP1-NFKB-dCas9 cell line and created two negative controls cell lines: a non-targeting sgRNA and an anti-GFP sgRNA. We designed 3 sgRNAs and created three CRISPRi-LincJADE1 cell lines and created three CRISPRi-LincANXA3 cell lines. We cloned the top 2 performing guides from our THP1 differentiation screen (10.1073/pnas.2322524121) and created 2 CRISPRi-GATA2AS1 cell lines and 2 CRISPRi-PPP2R5C-AS1 cell lines. RNA samples were collected at 0hrs and 24hrs after being treated with 100nM of PMA.