GSM9193677: Immortalized endometrial stromal cells transfected with pooled non-targeting siRNA and precipitated with rabbit IgG 3; Homo sapiens; OTHER
Targeted DNA amplicons were isolated using the Epicypher CUTANA Cut&Run Kit Version 3. A total of 550,000 cells per sample were collected in DNA LoBind 1.5mL tubes containing wash buffer. Cells were captured with BioMag Plus Concanavalin A beads and incubated with corresponding antibodies overnight at 4°C in antibody incubation buffer. After washing away antibodies, pAG-MNase generated by the NIEHS proteomics core was added and incubated for 30 minutes at room temperature. After washing, CaCl2 was added and incubated for 30 minutes in an aluminum cold block placed in ice. The protein-DNA complexes were released by incubating at 37°C for 10 minutes. DNA was purified using the Qiagen MiniElute PCR Purification Kit. Sequencing libraries were prepared using the NEBNext Ultra II DNA library preparation kit for Illumina according to the manufacturer's instructions, with 12-14 cycles of PCR amplification. The PCR products were quantified using the Qubit dsDNA HS Assay Kit, and quality control was performed with Tapestation Analysis. The libraries were sequenced by the NIEHS sequencing core.