GSM3660452: 4C_2.7kb_clone_replicate2; Mus musculus; OTHER
The initial steps of 4C protocol were performed as published before (Splinter et al., 2012; Methods (PMID: 22929766)). Briefly, cells are cross-linked using 2% formaldehyde for 10min at room temperature in PBS 10%FCS and nuclei are isolated, after which chromatin is digested with DpnII and subsequently ligated. After reversal of the cross-links, the DNA is purified and treated with the second restriction enzyme Csp6I. After a second re-ligation step, the sample is purified and a 4C PCR directly on this template for mouse genome viewpoints. In order to PCR amplify the ectopic TetO viewpoint, 4C templates were treated with Cas9 in vitro. Cas9 is targeted into the TetO repeats by using a sgRNA designed in between 4C viewpoint primers.
GEO Accession
GSM3660452