Daniel J. O'Connell, Raivo Kolde, Matthew Sooknah, Daniel B. Graham, Thomas B. Sundberg et al.
Reporter gene assays are a venerable tool for studying signaling pathways, but they lack the throughput and complexity necessary to contribute to a systems-level understanding of endogenous signaling networks. Here we present a parallel reporter assay, Transcription Factor activity sequencing (TF-seq), built on synthetic DNA enhancer elements, which enables parallel measurements in primary cells of the transcriptome and transcription factor activity from more than 40 signaling pathways. Using TF-seq in Myd88−/− macrophages, we captured dynamic pathway activity changes underpinning the global transcriptional changes of the innate immune response. We also applied TF-seq to investigate small-molecule mechanisms of action and find a role for NFκB activation and coordination of the STAT1 response in the macrophage reaction to the anti-inflammatory natural product halofuginone. Simultaneous TF-seq and global gene expression profiling presents an integrative approach for gaining mechanistic insight into pathway activity and transcriptional changes that result from genetic and small molecule perturbations.
Daniel J. O'Connell, Raivo Kolde, Matthew Sooknah, Daniel B. Graham, Thomas B. Sundberg, Isabel Latorre, Tarjei S. Mikkelsen, Ramnik J. Xavier. Simultaneous Pathway Activity Inference and Gene Expression Analysis Using RNA Sequencing. 2016-05-25. https://doi.org/10.1016/j.cels.2016.04.011
The 58-response-element lentiviral TF-seq library was assayed in wild-type mouse bone-marrow-derived macrophages after 30-minute pretreatment with vehicle or small molecules, followed by LPS stimulation; matched no-stimulation controls were included. Two 48-well plates provide four well positions per condition on each plate, yielding eight wells per condition.
The 58-response-element lentiviral TF-seq library was assayed in wild-type mouse bone-marrow-derived macrophages across a halofuginone dose series with or without 2 mM proline. Samples were collected at 0, 1, and 4 hours after LPS stimulation, with matched control samples and two replicate wells per dose arm.
A pooled 58-response-element lentiviral reporter library was assayed in wild-type and Myd88-knockout mouse bone-marrow-derived macrophages after 12 pathogen-associated molecular patterns at eight time points from 0 to 4 hours. The table contains the reporter RNA-UMI counts for 384 genotype/plate/well conditions, plus the deposited negative and spike-in controls.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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