4C of B16 CTCF pLoF Hmgcs1
4C-seq was performed as described previously (Krijger, P.H.L, 2020) with some modifications: 107 of cells were used for each point. The first restriction enzyme was CviQI (R0639, New England Biolabs, MA) and the second one was DpnII (R0543, New England Biolabs, MA). For the first round of two-step PCR library preparation procedure the following pair of primers was used; Reading primer: 5-TACACGACGCTCTTCCGATCTcacttggtccaaacagaaac-3; Non-reading primer: 5-ACTGGAGTTCAGACGTGTGCTCTTCCGATCTctctcattggctgaaagc-3. For the second round of PCR a common forward primer was used; 4C_Universal_Fw: 5- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCtacacgacgctcttccgatct-3 and the following reverse primers: WT cells repetitions 1-3: 5-CAAGCAGAAGACGGCATACGAGATCGTGATGTGactggagttcagacgtgtgct-3; 5- CAAGCAGAAGACGGCATACGAGATTGGTCAGTGactggagttcagacgtgtgct-3; 5-CAAGCAGAAGACGGCATACGAGATGATCTGGTGactggagttcagacgtgtgct-3; CTCF pLoF C13 repetitions 1-3: 5-CAAGCAGAAGACGGCATACGAGATACATCGGTGactggagttcagacgtgtgct-3; 5- CAAGCAGAAGACGGCATACGAGATCACTGTGTGactggagttcagacgtgtgct-3; 5- CAAGCAGAAGACGGCATACGAGATTCAAGTGTGactggagttcagacgtgtgct-3; CTCF pLoF C21 repetitions 1-3: 5- CAAGCAGAAGACGGCATACGAGATGCCTAAGTGactggagttcagacgtgtgct-3; 5- CAAGCAGAAGACGGCATACGAGATATTGGCGTGactggagttcagacgtgtgct-3; 5- CAAGCAGAAGACGGCATACGAGATCTGATCGTGactggagttcagacgtgtgct-3. NGS sequencing was done at the Technion Genome Center using an Illumina MiSeq system.