CTCF chromatin residence time controls 3D genome organization, gene expression and DNA methylation in pluripotent cells [ChIP-Seq, RNA-seq, Med-Seq]

The eleven zinc finger (ZF) protein CTCF regulates topologically associating domain (TAD) formation and transcription through selective binding to thousands of genomic sites. We replaced endogenous CTCF in mouse embryonic stem (ES) cells with GFP-tagged wildtype or mutant proteins lacking individual ZFs. We examined the various ES cell lines using next generation sequencing methods (ChIP-Seq, RNA-Seq, MedSeq, HiC) in order to identify additional determinants of CTCF positioning and function. Overall design: 1) ChIP-Seq of CTCF in mouse ES cells expressing GFP-CTCF or mutant proteins lacking individual ZFs 1, 8, 9, 10 or 11. Seven ChIP-Seq libraries were prepared using rabbit polyclonal antibodies against CTCF (home made). As control we used pre-immune serum. 2) RNA-Seq of mRNA derived from mouse ES cells expressing GFP-CTCF (GC) or a CTCF mutant protein lacking ZF8 (d8). RNA-Seq libraries were prepared from two GC lines (GC1, GC2) and three d8 lines (d8-1, d8-2, d8-3). Triplicate samples were isolated, except in the case of d8-3, where duplicates were isolated. 3) MedSeq of LpnPI-digested genomic DNA from mouse ES cells expressing GFP-CTCF (GC) or a CTCF mutant protein lacking ZF8 (del8). MedSeq libraries were prepared from two GC ES lines (GC1, GC2) and three del8 ES lines (d8-1, d8-2, d8-3). Two technical replicates were analyzed from each line.