Extended Data Fig. 2: Native ChIP-MS to identify CTCF colocalizing proteins.
a, FLAG-tag integrated at the C-terminus of CTCF via CRISPR genome editing. AH1, 2: arm of homology 1, 2, respectively. b, FLAG pull-down followed by CTCF western blot in benzonase solubilized nuclear pellet (NP) of ESCs. IP, immunoprecipitation. c, Native FLAG-CTCF immunoprecipitation in ESCs and MNs results in identification of known CTCF interactors and novel proteins. The mean peptide counts from two biological replicates of FLAG-CTCF immunoprecipitations were normalized to the control FLAG immunoprecipitation from untagged cells. Candidates filtered from the top of the list are shown (see Supplementary Dataset 4 for all). IP, immunoprecipitation. d, Venn diagram showing the overlap of CTCF-boundary related candidates identified in 4 independent (two for library A and two for library B) screens and CTCF ChIP-MS (native and crosslinked) approaches (see Supplementary Dataset 5 for overlapping candidates). P value cutoff = 0.05 for screens. See Supplementary Dataset 2 and Supplementary Dataset 4 for statistics in each screen and mass-spectrometry experiments. e, Western blot analysis of CTCF and MAZ in different cellular fractions in mESCs and MNs. CE: cytoplasmic extract, NE: nuclear extract, NP: nuclear pellet. f, Western blot analysis of CTCF, SMC1, and MAZ upon FLAG-CTCF immunoprecipitation from nuclear pellet of mESCs. IP, immunoprecipitation. g, Western blot analysis of FLAG, RAD21, and MAZ upon FLAG-CTCF immunoprecipitation from 293FT nuclear extract. IP, immunoprecipitation. h, Schematic of candidate selection from genetic and biochemical approaches for secondary loss-of-function screens.
