GSM8539064: HEK293T,EX,H3K9AC,5W,IN,rep2; Homo sapiens; OTHER
After the cells were treated according to the Cut-tag protocol, the DNA was extracted using DNA clean beads. For 5mC-CUT&Tag, pA/G-Tn5 assembled with Tn5-P5-mC was utilized to profile various histone modifications in K562, HEK-293T, and L1210 cells. This protocol was executed using the Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903), following the manufacturer's instructions. For 5mC-Multi-CUT&Tag, we employed anti-rabbit and anti-mouse nb-Tn5 assembled with Multi-P5-mC-1/2 to simultaneously profile multiple histone modifications in K562, HEK-293T, and L1210 cells. This process was performed using the Multi-CUT&Tag Library Prep Kit for Illumina (YEASEN, 12592ES), adhering to the provided guidelines. The 5mC-ATAC protocol was conducted in HEK-293T cells using Tn5 assembled with Tn5-P5-mC, facilitated by the ATAC-Seq Library Prep Kit for Illumina (Vazyme, TD711).  In each of the methods described above, the procedures were halted prior to library PCR amplification. At this stage, 10% of the library templates were reserved for direct 3' adaptor ligation (Vazyme, NE103) as the IN group (without bisulfite treatment). The remaining 90% of the library templates underwent bisulfite conversion (Zymo, D5030), followed by 3' adaptor ligation, constituting the BS group (with bisulfite treatment). Additionally, 1 ng of fragmented λDNA with methylated adaptors, prepared using the NEBNext Ultra II DNA Library Prep Kit (E7645S), was spiked in with the library templates before bisulfite conversion to monitor C-to-U conversion rates. Library PCR amplification was carried out using KAPA HiFi HotStart Uracil+ ReadyMix (KAPA, KK2801) with N5/D7 primers for 5mC-CUT&Tag and 5mC-ATAC, or Multi-P5/D7 primers for 5mC-Multi-CUT&Tag. DNA clean beads (Vazyme, N411) were employed for purification and size selection of the final library products.