GSM8532280: ChIP-seq on mutant CH12 cells, ectopically expressing Full-Length (FL)-CTCF (mutCH12_FLCTCF) with Snf2h Antibodies; Mus musculus; ChIP-Seq
MNase-ChIP-seq was performed on fixed cells using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, Cat #9003) following the manufacturer's protocol. Briefly, after obtaining MNase-digested lysates as described in the MNase-seq method, chromatin lysates were incubated with 5 μg of Histone H3 (Cell Signaling, #D2B12) XP® Rabbit mAb and rotated overnight at 4°C. Following this incubation, 30 µL of Protein G Magnetic Beads were added to the mixture and rotated for 2 hours at 4°C. The Protein G magnetic beads were then pelleted using a magnetic separation rack for 1–2 minutes. The supernatant was carefully removed, and the beads were washed three times with 1 mL of low salt wash buffer, incubating each wash for 5 minutes at 4°C with rotation. After the final low salt wash, 1 mL of high salt wash buffer was added, and the beads were incubated again for 5 minutes at 4°C with rotation. The beads were pelleted, and the supernatant was removed. Next, 150 µL of 1X ChIP Elution Buffer was added, and the chromatin was eluted from the antibody/Protein G magnetic beads by placing the tubes on a thermomixer at 65°C for 30 minutes with gentle shaking (1,000 rpm). The eluted chromatin supernatant was transferred to a new tube, and the immunoprecipitated DNA was purified according to the kit protocol. Sequencing libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (Illumina, Inc., USA). ChIP-seq protocol: 2x106 asynchronously growing cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine for 10 min, washed twice with 1×phosphate buffered saline (PBS), and resuspended in chromatin immunoprecipitation (ChIP) lysis buffer (150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 20 mM Tris–HCl pH8.0, 2 mM EDTA). Chromatin was sheared to 200–600 bp and immunoprecipitated with 5 μg of antibody. Immunoprecipitated DNA was purified with QIAquick columns (ZymaResearch). ATAC-seq protocol: CH12  cells (5 × 104) were pelleted by centrifugation and resuspended in 50 µL of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), gently pipetting up and down 10 times. Nuclei were pelleted by centrifugation at 2500g for 10 min at 4°C and resuspended in 25 µL of 2x TD Buffer (Illumina Cat #FC-121-1030), containing 8 µL of Tn5 Transposes (Illumina Cat #FC-121-1030) and 17 µL of nuclease free water. The rection was incubated for 30 min at 37°C. Tagmented DNA was purified by using a Zymo Research DNA Clean & Concentrator™-5 - Capped Columns. MeDIP-seq protocol: Genomic DNA (gDNA) was extracted from freshly growing CH12 cells and sonicated using a Covaris ultrasonicator to obtain a mean fragment size of 200 bp. For library preparation, 5 μg of sonicated gDNA was end-repaired and ligated with adaptors using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA). The reaction mixture was then purified with AMPure XP beads (Beckman-Coulter, CA, USA). For precise size selection, half of the sample was run on a 1.5% agarose gel, and DNA fragments of 150-250 bp were extracted from the gel. Both the size-selected and non-size-selected libraries were denatured at 95°C for 10 minutes, followed by incubation on ice for 10 minutes. The denatured DNA was subjected to immunoprecipitation with 5-Methylcytosine antibody (Catalog #: 61479, Active Motif) by incubating the reaction mixture at 4°C for 15 hours. The immunoprecipitated DNA was size-selected again using AMPure XP beads and amplified using Q5 High-Fidelity DNA Polymerase (NEB, M0491). The amplification products were purified with AMPure XP beads. Finally, the purity and fragment length distribution were assessed using a Bioanalyzer 2100 (Agilent Technologies), and the libraries were subjected to deep sequencing on the Illumina HiSeq 2000 platform. MNase-ChIP-seq and MeDIP-seq libraries were prepared using  NEBNext® Ultra™ II DNA Library Prep Kit (Illumina, Inc., USA) ChIP-seq libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina, Inc., USA) or NEBNext UltraII DNA Library Prep Kit for Illumina (BioLabs, catalog # E7645S).  Libraries were amplified using 15 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. ATAC-seq libraries were amplified using NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs). The libraries were size-selected  by adding 1.8X volume Agencourt AMPure XP beads (Beckman, 63881). ChIP-seq libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina, Inc., USA) or NEBNext UltraII DNA Library Prep Kit for Illumina (BioLabs, catalog # E7645S).  Libraries were amplified using 15 cycles on the thermocycler.