ChIP-seq Protocol for Chromatin Fragmentation

4×10⁷ cells were washed in PBS and cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched by addition of glycine (125 mM final concentration) for 5 min. For Nuclei isolation, cells were resuspended in cell lysis buffer (50 mM Tris pH8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), incubated the tube on ice for 20 min to swell. Harvested the nuclei by centrifugation at 2000g for 5 min at 4 °C resuspended in 1 ml ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.0) and incubated on ice for 10 min. Chromatin was fragmented to 200–500 bp using 12 cycles using the Vibra-Cell Ultrasonic Liquid Processors (SONICS, Newtown, CT, USA). For each IP, chromatin was immunoprecipitated with 2 mg of antibody in IP dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) at 4 °C overnight. Chromatin was precleared for 2 h each with protein G agarose beads (Cell Signaling Technology, Danvers, MA, USA) before immunoprecipitation. The immunoprecipitated material was washed, once in TSE I buffer (20 mM TrisHCl pH 8.0, 2 mM EDTA pH8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), once in TSE II buffer (20 mM TrisHCl pH 8.0, 2 mM EDTA pH8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), once in LiCl buffer (10 mM TrisHCl pH 8.0, 250 mM LiCl, 1% deoxycholic acid, 1% NP40) and once in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH8.0) before elution in elution buffer (100 mM NaHCO3, 1% SDS). Antibodies used in this study were listed in the Supplementary Information. The samples were removed from beads, reversed cross-linked overnight at 65 °C and DNA was isolated using QIAquick PCR Purification Kit (Germantown, MD, USA). Precipitated DNA was analyzed by high-throughput sequencing (Beijing Genomics Institute, Beijing, China).

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Li X., Wang S., Xie Y., Jiang H., Guo J., Wang Y., Peng Z., Hu M., Wang M., Wang J., Li Q., Wang Y, & Liu Z. (2023). Deacetylation induced nuclear condensation of HP1γ promotes multiple myeloma drug resistance. Nature Communications, 14, 1290.

Publication 2023

Agarose Antibodies Antibody Buffer Cell Centrifugation Chip Chromatin Deoxycholic acid Dilution Edta Formaldehyde Glycerol Glycine Immunoprecipitation Isolation Nacl Nahco3 Np 40 Nuclei Protein g Tris Triton x 100 Ultrasonic

Corresponding Organization :

Other organizations : Tianjin Medical University, Tianjin Medical University Cancer Institute and Hospital

Top 5 similar protocols

Variable analysis

independent variables

Cross-linking with 1% formaldehyde for 10 min at room temperature
Quenching with glycine (125 mM final concentration) for 5 min
Chromatin fragmentation to 200-500 bp using 12 cycles of sonication
Immunoprecipitation with 2 mg of antibody

dependent variables

Precipitated DNA analyzed by high-throughput sequencing

control variables

Cell lysis buffer (50 mM Tris pH8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100)
ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.0)
IP dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0)
TSE I buffer (20 mM TrisHCl pH 8.0, 2 mM EDTA pH8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS)
TSE II buffer (20 mM TrisHCl pH 8.0, 2 mM EDTA pH8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS)
LiCl buffer (10 mM TrisHCl pH 8.0, 250 mM LiCl, 1% deoxycholic acid, 1% NP40)
TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH8.0)
Elution buffer (100 mM NaHCO3, 1% SDS)

controls

Positive control: Not mentioned
Negative control: Not mentioned

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