GSM4978051: CTCF-ChIP-D3-shLuci-Thy1+ [ChIP-seq]; Mus musculus; ChIP-Seq
ChIP experiments were performed as previously described. Briefly, 1×10e7 cells were crosslinked with 1% formaldehyde at room temperature for 10 min. Then the reaction was stopped by adding glycine (final concentration, 0.125 M). Crosslinked cells were lysed in ChIP SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)) containing 1 X Protease Inhibitor Cocktail and PMSF, then sonicated to achieve a chromatin sized of 200-400 bp. After sonication, the supernatant was diluted with IP buffer and then co-incubated with antibodies–Dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Immune complexes were washed with the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)) and TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Antibody-bound chromatin was reverse-crosslinked, and the ChIPed DNA samples were purified for ChIP-Seq library generation. The ChIPmentation experiments were performed as previously described. Briefly, 1×10e5 cells were crosslinked with 1% formaldehyde at room temperature for 10 min. Then the reaction was stopped by adding glycine (final concentration, 0.125 M). Crosslinked cells were lysed in lysis buffer (10 mM Tris-HCl,100 mM NaCl (pH 8.0),1 mM EDTA, 0.5 mM EGTA,0.1% NaDeoxycholate, 0.5% N-lauroylsarcosine, 1 x protease inhibitors), then sonicated to achieve a chromatin sized of 200-700 bp.Triton X-100 was added to a final concentration of 1%. After centrifuge, the supernatant was co-incubated with antibodies–Dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Immune complexes were washed with the following buffers: low salt wash buffer, high salt wash buffer, LiCl wash buffer, TE buffer and 10 mM Tris-HCl (pH 8.0). For tagmentation, bead bound chromatin was resuspended in 45 μl of tagmentation buffer, 5 μl of transposase (Vazyme) was added and samples were incubated at 37°C for 10 min followed by two washes with low-salt buffer. Antibody-bound chromatin was reverse-crosslinked, and the ChIPed DNA samples were purified for ChIP-seq library generation. ChIP-seq sequencing libraries were constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2.