GSM8077765: HCT116_PCHiC_Nut1h-Rad21_BR2; Homo sapiens; OTHER
Hi-C: Cells were fixed in DMEM medium supplemented with 10% FBS and 2% methanol-free formaldehyde for 10 minutes rotating at room temperature. After quenching the formaldehyde with 0.125M glycine and washing the cells with 1X PBS, nuclei were extracted by lysing the cells in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA630 (SIGMA-ALDRICH #18896-50ML), 1× cOmplete EDTA-free protease inhibitor cocktail. Chromatin was digested overnight with HindIII enzyme and the cohesive restriction fragment ends were filled-in with dCTT, dTTP, dGTP and biotin-14-dATP(Invitrogen #19524-016) nucleotides. After blunt-end ligating the restriction fragment ends, the chromatin was decrosslinked by incubating overnight with proteinase K and purified by phenol:chloroform:isoamyl alcohol 25:24:1 (SIGMA-ALDRICH #P3803-100ML) extraction.Non-informative biotin at restriction fragment ends was removed by incubating the samples with dATP and T4 DNA polymerase (New England Biolabs#M0203S). After purifying the DNA again with a phenol:chloroform:isoamyl alcohol extraction, 10µg of the chromatin was sheared using a Covaris M220 focused ultrasonicator in 130µl cuvettes using the following parameters: 20% duty factor, 50 peak incident power, 200 cycles per burst, 65 seconds. The ends were end-repaired and dATP-tailed, followed by a biotin-pulldown using Dynabeads MyOne streptavidin C1 paramagnetic beads (Thermo Fisher #65001) to enrich for those DNA fragments which contain information of a chromatin loop. PE Illumina adapters(Supplementary Data 9) were ligated to the sample and the library was amplified for 8 cycles. Finally, the library was purified using a SPRI bead double-sided selection (0.4-1 volumes). Size and concentration of the finished Hi-C libraries was assessed by DNA ScreenTape Analysis (Agilent #5067-5582) on an Agilent 2200 Tapestation. PCHiC: Cells were fixed in DMEM medium supplemented with 10% FBS and 2% methanol-free formaldehyde for 10 minutes rotating at room temperature. After quenching the formaldehyde with 0.125M glycine and washing the cells with 1X PBS, nuclei were extracted by lysing the cells in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA630 (SIGMA-ALDRICH #18896-50ML), 1× cOmplete EDTA-free protease inhibitor cocktail. Chromatin was digested overnight with HindIII enzyme and the cohesive restriction fragment ends were filled-in with dCTT, dTTP, dGTP and biotin-14-dATP(Invitrogen #19524-016) nucleotides. After blunt-end ligating the restriction fragment ends, the chromatin was decrosslinked by incubating overnight with proteinase K and purified by phenol:chloroform:isoamyl alcohol 25:24:1 (SIGMA-ALDRICH #P3803-100ML) extraction.Non-informative biotin at restriction fragment ends was removed by incubating the samples with dATP and T4 DNA polymerase (New England Biolabs#M0203S). After purifying the DNA again with a phenol:chloroform:isoamyl alcohol extraction, 10µg of the chromatin was sheared using a Covaris M220 focused ultrasonicator in 130µl cuvettes using the following parameters: 20% duty factor, 50 peak incident power, 200 cycles per burst, 65 seconds. The ends were end-repaired and dATP-tailed, followed by a biotin-pulldown using Dynabeads MyOne streptavidin C1 paramagnetic beads (Thermo Fisher #65001) to enrich for those DNA fragments which contain information of a chromatin loop. PE Illumina adapters(Supplementary Data 9) were ligated to the sample and the library was amplified for 8 cycles. Finally, the library was purified using a SPRI bead double-sided selection (0.4-1 volumes). Size and concentration of the finished Hi-C libraries was assessed by DNA ScreenTape Analysis (Agilent #5067-5582) on an Agilent 2200 Tapestation. PCHi-C: libraries, 500-1000ng of Hi-C library were captured using the SureSelectXT Target Enrichment System for the Illumina Platform (Agilent Technologies) as instructed by the manufacturer. Captured library was amplified a total of 4 cycles, and the size and concentration of the finished PCHi-C libraries was assessed by high-sensitivity DNA ScreenTape Analysis (Agilent #5067-5584) on an Agilent 2200 Tapestation.