GSM7882467: hTSC-BL-CEBPB, ChIP-seq, rep1; Homo sapiens; ChIP-Seq
For ChIP-seq sample extraction, hTSCs-BL and STB-BL cells were dissociated with TrypLE for 8 min at 37℃ and washed with phosphate buffer solution for 3 times. About 5 × 106 cells were used for cross-linking by 1% formaldehyde. After rotating at room temperature for 10 min, the crosslinking was quenched by 125 mM Glycine and rotated for 10 min at room temperature. The crosslinked cells were quick-froze with liquid N2. For CUT&Tag sample extraction, STB-BL cells were dissociated with TrypLE for 8 min at 37℃ and washed with phosphate buffer solution. About 5 × 104 cells were resuspend with wash buffer. The experiment of ChIP-seq was performed as previously described (A selective WDR5 degrader inhibits acute myeloid leukemia in patient-derived mouse models). Briefly, about 5 × 106 hTSCs-BL and STB-BL were used for cross-linking by 1% formaldehyde and quenched by 125 mM glycine. Cells were lysed and nuclei were sonicated and diluted to a final concentration of 0.1% by SDS. Protein A/G magnetic beads were added and incubated at 4°C for 1 h. Pre-cleared chromatin was incubated with CEBPB antibody (Cat#23431-1-AP, Proteintech) or monoclonal rabbit isotype control (Cat#ab172730, Abcam) overnight at 4°C. Then Protein A/G magnetic beads were added to capture the specific chromatin complexes. The supernatants were collected with 500 mM NaCl and incubated at 65 °C overnight for reverse crosslinking. Sample was incubated at 37°C for 1 h with RNase A, and was incubated at 50°C for 1 h with proteinase-K. The released DNA was purified by the QIAquick PCR purification Kit and processed to sequencing library preparation. Multiplexed ChIP-seq libraries were prepared by NEBNext Ultra II DNA Library Prep Kit (Cat#E7645S, NEB). The final libraries and multiplexed libraries were subjected to Illumina Sequencing 2500 platforms.For CUT&Tag,the experiment was conducted according to the instructions provided by Vazyme (Cat#TD904). In brief, approximately 5 × 104 cells were harvested and counted, then mixed with activated ConA Beads and incubated overnight with primary antibodies (CEBPB, Cat#23431-1-AP, Proteintech and FOSL2, Cat#351814, USBiological). The secondary antibody (Cat#Ab207, Vazyme) was incubated at room temperature (RT) with rotation for 1 hour. After removing the supernatant, pA/G-Tnp Pro and TTBL were separately incubated with rotation at RT and 37℃ for 1 hour. The supernatant was then mixed with DNA Extract Beads Pro to dissolve DNA with ddH2O and used directly for PCR amplification. Subsequently, VAHTS DNA Clean Beads were added to purify the DNA, which was then subjected to next-generation sequencing using the Illumina NovaSeq 6000 platform.