Chromatin looping evidence between PAX3-FOXO1 bound super enhancer and MYOD1 promoter [4C-seq]

Circular chromatin confirmation capture in rhabdomyosarcoma cells was performed with viewpoints at the PAX3-FOXO1 bound super enhancer upstream of MYOD1, and also at the MYOD1 promoter, to find support for looping between these two genetic elements almost 80 thousand base pairs apart. 4C-seq was performed on fixed cells, which were exposed to 4-bp cutter DpnII for the primary restriction enzyme, re-ligated in dilute conditions, followed by reversal of crosslinks and purification. Csp6I was used to reduce template to sizes amenable to inverse PCR for viewpoint amplification. 4C samples of RMS cell line (RH4) were amplified at anchors within the viewpoint.