Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
Blood is generated by a constant stream of differentiating haematopoietic progenitor cells. The process is controlled by an immensely complex gene regulatory networks. These have been difficult to comprehend using correlative evidence and limited systematic functional data. Hoxb8-FL cell line is a model system of lympho-myeloid progenitors, which self-renews in vitro and is amenable to genetic perturbations. To construct a functionally defined transcription factor (TF) network we targeted 39 transcription factors using CRISPR/Cas9 gene targeting in Hoxb8-FL cells. We measured the resulting transcriptional changes by small scale RNA-Seq within 2-4 d of each perturbation. Our network analysis revealed >17,000 TF-target interactions across >7,000 target genes, established new interactions among TFs and shed new light on the mechanisms maintaining self-renewal and multipotency.
Overall design
RNA-Seq profiles of Hoxb8-FL cells with CRISPR/Cas9 perturbations against 39 TFs. Samples are organised in plates as indicated, each plate contains samples corresponding to 3 targeted TFs, each with 3 sgRNAs and 2-3 sets of controls (either non-targeting sgRNA or sgRNA targeting the Rosa26 locus). Each condition was performed in 8 replicates. Additionally, 8 replicates were added for Hoxb8-FL cells 16 h after switching off Hoxb8 expression. Additionally, we provide ChIP data for 16 TFs and H3K27Ac histome modification.
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