GSM5930797: K562 e4i rep2 [Trac-looping]; Homo sapiens; OTHER
Modified Trac-looping was performed for mapping chromatin architecture in 4 conditions (wildtype, perturbation of individual e3 or e7, and perturbation of both e3&e7). One million cells were fixed with 1% formaldehyde in the culture medium at room temperature for 10 minutes. Cells were washed with PBS and resuspended with a reaction buffer (50mM Tris acetate, pH 7.5, 150mM potassium acetate, 10mM magnesium acetate, 4mM spermidine, 0.5% NP-40). The short adapter and biotinylated bridging linker with Tn5, and then incubated at room temperature for 20 minutes to generate Tnp bridging complex. This complex was added to cells and mixed gently. Chromatin interacting regions were bridged at this step. EDTA was added to stop the reaction and reverse crosslinked by incubating at 65°C overnight. Genomic DNA was extracted by Phenol-Chloroform and the purified DNA was digested with restriction enzymes MluCI/NlaIII. Streptavidin beads were used to enrich biotin-labeled DNA fragments, followed by adapter ligation and amplification by PCR with Illumina multiplexing indexed primers. Libraries were purified and submitted for paired-end sequencing (Illumina NovaSeq 6000). The detailed protocol is being prepared for publishing elsewhere.