O'Connell, D.J. et al. Cell Systems 2, 323–334 (2016).

Reporter genes are common tools for revealing transcriptional states, regulatory circuitry and responses to stimuli. To scale up to study the activation of multiple pathways, O'Connell et al. switched to sequencing rather than a light- or color-based reporter readout. The researchers generated lentiviral vectors, each encoding synthetic DNA response elements for one of 40 signaling pathways with a corresponding sequence tag. They then transfected cells as a pool and used RNA sequencing to measure tag frequencies as well as global transcription. Using this transcription factor activity sequencing (TF-seq) approach, O'Connell et al. inferred pathway dynamics in bone-marrow-derived macrophages from Myd88-knockout mice upon stimulation by small molecules or pathogen-associated molecular pattern molecules (PAMPs). TF-seq pathway activity measurements could not be reproduced using RNA or ChIP sequencing data alone.