GSM6213812: K562 Micro-C, bio rep7; Homo sapiens; OTHER
K562 cells were first crosslinked with 1% formaldehyde (Sigma, 47608) for 10 min at room temperature and then quenched by 0.75 M Tris pH 7.5. After two rounds of PBS washes, cells were further crosslinked with 3mM DSG (Santa Cruz, sc-285455A) for 45 min at room temperature. After quenching with 0.75 M Tris pH 7.5 and the following two rounds of PBS washes, K562 cell pellets were snap frozen in liquid nitrogen and stored at -80 °C. Micro-C libraries were prepared following the procedure as previously described (Hsieh et al., Mol Cell, 2020). In brief, 5 million of K562 cell pellets were treated in lysis buffer containing 0.2% NP-40. The extracted nuclei were then digested by 1,000 Gel Units of MNase (NEB, M0247S) at 37°C for 10 min with 80 to 90% of fragmented chromatin were mononucleosomes. The MNase-digested nucleosomes were then end repaired and incorporated with Biotin-14-dATP (Jena Bioscience, NU-835-BIO14-L) and Biotin-11-dCTP (Jena Bioscience, NU-809-BIOX-L). Then the nucleosomes were ligated by T4 DNA Ligase (NEB, M0202) and unligated products were removed by exonuclease III (NEB, M0206). The de-crosslinked DNA were purified by phenol/chloroform/isoamyl alcohol and separated on 6% PAGE gel. A band at the size of 250 to 400 bp were excised and DNA was extracted and then purified by MyOne™ Streptavidin C1 (Thermo Fisher Scientific, 65001). For the library preparation, end-repair/A-tailing, and adaptor ligation were performed on-bead using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607-01). Ligated DNA were then amplified using 12 cycles on the thermocycler and libraries were purified using the VAHTS DNA Clean Beads (Vazyme, N411-01). Micro-C libraries were sequenced paired-end 150 bp with Illumina Novaseq 6000 sequencer.