Identification of TBX5 Binding Sites in Mouse Embryonic Forelimbs

In order to identify putative regulatory targets of the TBX5 transcription factor, ChIP-Seq was performed on chromatin isolated from E10.5 mouse embryonic forelimb buds. Overall design: ChIP-seq of mouse embryonic day 10.5 forelimbs against TBX5. Roughly 50 µg of sheared chromatin was isolated from E10.5 forelimb buds from outbred ICR mice (Envigo) and incubated with a TBX5 polyclonal antibody (R&D Systems AF5918). After isolation of antibody bound chromatin fragments with a Protein G Agarose Columns (Active Motif), libraries were prepared using the NEBNext Ultra II Library Prep Kit and amplified for 15 PCR cycles. Sera-Mag SpeedBeads were used to perform size selection to remove primer and adapter fragments.