SCA-seq of wild-type HEK293T
Cell cultureDerivative human cell line which expresses a mutant version of the SV40 large T antigen (HEK 293T) [abclonal] and CTCF allele-specific knockout 293T cell line [abclonal] were each maintained in DMEM-high glucose [Thermo Fisher 11995065] supplemented with 10% fetal bovine serum (FBS) [Thermo fisher 1009141]. The CTCF +/- cell line was purchased from ABclonal, and validated by genotyping, western blot, qPCR and RNA-seq (Sfig14). Cross-linking5 million cells were washed 1 time in chilled 1X phosphate buffered saline (PBS) in a 15 mL centrifuge tube, pelleted by centrifugation at 500xg for 3 min at 4. Cells were resuspended by gently pipetting in 5 mL 1X PBS with formaladehyde (1% final concentration). Incubating cells at room temperature for 10 min, add 265 L of 2.5 M glycine (125 mM final concentration) and incubate at room temperature for 5 min to quench the cross-linking. Centrifugate the mix at 500xg for 3 min at 4. Wash cells 2 times with chilled 1X PBS. Nuclei isolation and methylationCell pellet was resuspended with cold lysis buffer: 10 mM HEPES-NaOH pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1X proteinase inhibitor [Sigma 11873580001], 0.1% Tween-20, 0.1 mg/ml BSA, 0.1 mM EDTA, 0.5% CA-630, incubate on ice for 5 min. Centrifugate lysis mixure at 500xg for 5 min at 4 to collect the nuclei. Washed the nuclei once with 1X GC buffer [NEB M0227L] then resuspend 2 million nuclei in 500 L methylation reaction mixture: 1X GC buffer, 200 U M. CvipI [NEB M0227L], 96 M S-adenosylmethionine, 300 mM Sucrose, 0.1 mg BSA, 1X proteinase inhibitor, 0.1% Tween-20. Incubate the reaction for 3 hours at 37, add 96 M SAM and 20 U M.CvipI per hour. Centrifugate at 500xg for 10 min at 4 to collect nuclei, wash the nuclei once with chilled HEPES-NaOH pH7.5 and centrifugate to collect nuclei. Restriction enzyme digestResuspend nuclei with 81 L cold HEPES-NaOH pH7.5, add 9 L 1% SDS and react at 65 for 10 min to denature the chromatin, take the tube on ice immediately after reaction. Add 5 L 20% Triton X-100 and incubate on ice for 10 min to quench SDS. Prepare digestion mixture: 140 U DpnII [NEB R0543L], 14 L 10X HEPES-buffer3.1 [50 mM HEPES-NaOH pH 8.0, 100 mM NaCl, 10 mM MgCl2, 100 g/mL BSA], add nuclei suspension and nuclease-free water into mixture to achieve a final volume of 140 L. Incubate digest mixture in a thermomixer at 37 for 18 hours with 900 rpm rotation. LigationDpnII digests were heat inactivated at 65 for 20 min with 700 rpm rotation, average digests to 70 L per tube, add 14 L T4 DNA Ligase buffer [NEB M0202L], 14 L T4 DNA Ligase [NEB M0202L], 1 mM ATP and nuclease-free water to achieve a final volume of 140 L. The ligation was incubated at 16 for 10 hours with 800 rpm rotation. Reverse cross-linking and DNA purificationCollect all ligation into one 1.5 mL tube, add equal volume of 2X sera-lysis [2% Polyvinylpyrrolidone 40, 2% Sodium metabisulfite, 1.0 M Sodium Chloride, 0.2 M Tris-HCl pH 8.0, 0.1 M EDTA, 2.5% SDS], add 5 L RNaseA [QIAGEN 19101], incubate at 56 for 30 min. Add 10 L Proteinase K [QIAGEN 19131], 50 overnight incubation with 900 rpm rotation. DNA was purified with high molecular weight gDNA extraction protocol [Baptiste Mayjonade, 2016].