GSM8486585: Endothelial cells, miR-NC, IgG; Homo sapiens; OTHER
The CUT&Tag assay used the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903). Library construction was performed according to the CUT&Tag library construction protocol with some modifications[1]. Cells were crosslinked with 1 % formaldehyde for 10 min at room temperature and quenched with 125 mM glycine, this step was optional. Concanavalin A coated magnetic activated beads were added per sample and incubated at RT for 15 min. The binding cells to beads were pre-cleared and immunoprecipitated with anti-NIPBL and anti H3K27AC antibodies. For increasing quantity of Protein A binding sites, secondary antibody were employed. After washing, the cells were treated with PA/G-Tn5 adapter complex containing 10mM MnCl2 to get CUT&Tag fragments DNA libraries. The DNA libraries were amplified for 15 cycles and sequenced using Illumina NovaSeq 6000 with paired-end 150 as the sequencing mode.