GSM9409167: First ChIP_in_WT_Day2 overexpressing HOXA1-HA; Homo sapiens; ChIP-Seq
7-8 x 10^6 Day 2 differentiated cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature, then quenched with 0.125 M glycine for 5 minutes. Cells were washed three times with cold PBS and harvested in DNA LoBind Tubes (Eppendorf, 0030108035/0030108051). Sonication was performed using a Bioruptor (Diagenode, B01020001) on high setting (30 sec on, 30 sec off) for 30 minutes in high salt lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.05% SDS, 1% Triton X-100) containing a protease inhibitor cocktail. Chromatin samples were precleared with 15 μL each of Dynabeads Protein G and Protein A, and rotated at 4°C for 1 hour. Precleared samples were then incubated with 4 µg of specific antibody at 4°C for 16 hours. After antibody binding, 15 μL each of Dynabeads Protein G and Protein A were added and incubated for an additional 2 hours at 4°C. The chromatin-antibody complexes underwent four washes with high salt buffer, two washes with low salt buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate), and one wash with TE buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 50 mM NaCl).Complexes were eluted in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) on a Thermal Mixer (Thermo, 13687712) at 65°C for 4 hours, followed by treatment with 10 µg RNase and 10 µg Proteinase K at 55°C for 1 hour on the Thermal Mixer. DNA was purified using a QIAquick PCR Purification Kit (QIAGEN, 28006). For Re-ChIP assays, the starting material was scaled to 2 x 107 cells. Chromatin was first immunoprecipitated with 12 µg of anti-HA antibody and competitively eluted under native conditions using HA peptide (1 mg/mL) (MCE, HY-P0239). Subsequently, 30% of the eluate was retained as the primary ChIP fraction, while the remaining 70% was diluted and subjected to incubation with 12 µg of anti-NONO antibody overnight, and then and captured with Dynabeads. The purified DNA library preparation was constructed using VAHTS Universal Plus DNA Library Prep Kit for Illumina (Vazyme, ND617) in accordance with the manufacturer's instruction. Libraries were sequenced by the Haplox company (Jiangxi, China) using Illumina NovaSeq 6000. The purified DNA library preparation was constructed using VAHTS Universal Plus DNA Library Prep Kit for Illumina (Vazyme, ND617) in accordance with the manufacturer's instruction.