Meg3 binding sites in mouse insulinoma cells

We have shown that increased β-cell proliferation in functioning pancreatic neuroendocrine tumors (insulinomas) correlated with reduced expression of the long non-coding RNA Meg3 and increased expression of the oncogenic receptor c-Met. To investigate the target binding sites of Meg3 in and around the c-Met gene, we did ChIRP-Seq using biotinylated probes from the mouse Meg3 RNA sequence. This would help us better understand how Meg3 regulates ithe expression of c-Met to control β-cell proliferation in insulinoma cells. Glutaraldehyde cross-linked chromatin was prepared from 10 million cells of a mouse pancreatic islet β-cell line (MIN6-4N) stably transfected with Vector or stably overexpressing Meg3. Chromatin isolation by RNA purification (ChIRP) as performed using 2 pools of odd-numbered or even-numbered biotinylated probes from the mouse Meg3 RNA sequence using standard protocols (Sigma). ChIRP-Seq libraries (input, odd probe and even probe) were prepared with Diagenode's MicroPlex Library Preparation kit and IP-Star Compact protocol. ChIRP-Seq libraries were sequenced for single-end 50 bp, and sequences were analyzed (NIDDK genomics core facility and Genomatix).