GSM8844137: 22Rv1_NOMe-HiC_1; Homo sapiens; OTHER
Cells were harvested, crosslinked, and lysed to isolate intact nuclei. Methyl-Micro-C library: Nuclei were treated with MNase and proximity ligation was performed with a biotinylated bridge. Ligated DNA products underwent end preparation (NEB, E7546S) and methylated adapters (NEB, E7165AAVIAL) are added. After enriching ligated products, oxidation of methylated and hydroxy-methylated cytosines and deamination of unprotected unmethylated cytosines were performed using EM-seq conversion module (NEB, E7125S). Methyl-Micro-C library was generated by performing indexing PCR and sequenced using Illumina NovaSeq sequencing systems, following the guidelines of the manufacturer. NOMe-HiC library: After reaching 70~80 confluence, 22Rv1 cells were detached with 0.5% Trypsin and harvested by centrifuge at 300g for 5min. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature, then quenched by adding 0.2M Glycine. After washing with ice-cold PBS, cells were suspended in the nuclei isolation buffer with RNase inhibitors and incubated on ice for 1hr. Subsequent to nuclei membrane permineralization, chromatin was digested using 100U DpnII (NEB, R0543L) overnight at 37 ℃ with RNase inhibitors. The next day, DpnII was inactivated, and nuclei suspension was cooled to room temperature and subjected to biotin fill-in. Proximal segments were in situ ligated using T4 DNA ligase (NEB, M0202) supplemented with RNase inhibitors. Then GpC methyltransferase footprint was performed by incubating the nuclei with M.CviPI (NEB, M0227L), and PBS was added to stop the reaction. The nuclei were reverse crosslinked, DNA was precipitated and sonicated to average 400bp followed by 1xAMPure beads cleanup. Biotin pulldown was performed using streptavidin beads, DNA on beads was end-repaired, d-A tailed, and ligated with EM-seq adaptor (NEB). Bisulfite conversion was conducted using the EZ DNA Methylation-Gold Kit (D5006). Bisulfite converted DNA library was amplified using NEB EM-seq primers and KAPA HiFi Uracil+ ReadyMix (Roche, KK2801).