5hmC-seq in human MonoMac6 acute myeloid leukemia (AML) cells with and without knockdown of TET1

To dissect the mechanism underlying the regulation of TET1 on its target genes in AML, we performed 5hmC-sequencing for genomic DNA isolated from human MonoMac6 AML cells with and without knockdown of TET1. Overall design: We lentivirally transduced pLKO.1-based lentiviral shRNA targeting TET1 (i.e., shTET1-1, and shTET1-2) or scramble shRNA (i.e., shNS) into MonoMac6 cells. After puromycin selection (1 µg/mL) for two passages, cells were collected and genomic DNA was extracted using DNeasy blood and tissue kit (Qiagen). DNA was fragmented and labelled with 6-N3-glucose using ß-GT enzyme. Biotin tags were added onto the 6-N3-glucose with Huisgen cycloaddition (click) chemistry reaction using DBCO-PEG4-biotin. The biotin-labelled 5hmC containing DNA was enriched with streptavidin-coupled magnetic beads. The enriched DNA was used for library preparation and next-generation sequencing.