GSM9376488: CUTTag_REXO4_siYTHDF2_Rep1; Homo sapiens; OTHER
The CUT&Tag procedure was conducted following a previously established protocol, with some alterations detailed below. In brief, 1 × 105 cells were subjected to centrifugation (600 g for 3 min) at room temperature, washed twice with 500 l of wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and 1 × protease inhibitor), and then resuspended in 100 l of wash buffer within low-retention PCR tubes. Concanavalin-A-coated magnetic beads (Vazyme) were pre-activated and resuspended in an equivalent volume of binding buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1 mM CaCl2, and 1 mM MnCl2). A total of 10 l of these activated beads was mixed with the cells and incubated gently for 10 min with rotation.  The cells bound to the beads were magnetically separated to discard the supernatant, washed three times with binding buffer, and resuspended in 50 l of antibody buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitor, 0.05% digitonin, 0.01% NP-40, and 2 mM EDTA). Subsequently, 1 g of primary antibodies was added, and the mixture was incubated overnight at 4 ºC. As controls, 2 U of RNase H (NEB) was included during the antibody incubation phase, while rabbit or mouse IgG served as the IgG control. Secondary antibodies (Vazyme) were diluted at a ratio of 1:100 in dig-wash buffer (5% digitonin in wash buffer) followed by rotation for 1 hr at room temperature to facilitate antibody binding. The bead-bound cells underwent three washes with dig-wash buffer to eliminate unbound antibodies. The pAG-Tn5 adapter complex (Vazyme) was then prepared in dig-300 buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitors, 0.01% digitonin, and 0.01% NP-40) to achieve a final concentration of 0.2 M. The cells bound to the beads were resuspended in 100 l of the pAG-Tn5 mix and incubated for 1 hr at room temperature before removing the supernatant. Following thorough washing, the tagmentation reaction was carried out in 50 l of tagmentation buffer (10 mM TAPS-KOH, pH 8.3, 10 mM MgCl2, and 10% DMF) at 37 ºC for 1 hr. Afterward, 2 µl of 10% SDS and 0.3 pg of spike-in DNA were added, and the mixture was incubated for 15 min at 55 ºC. DNA purification was then performed using DNA Extract Beads Pro (Vazyme). This leverages the purification tag on the sequencing adaptor, which is transposed by Tn5 and binds specifically to the beads. Following binding and washing steps, the DNA was resuspended in 15 l of ddH2O. For the strand displacement reaction, the eluent was mixed with 10 U of Bst 3.0 WarmStart DNA polymerase (NEB) in 1 × NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB) at 72 °C for 10 min and halted by incubation at 95 ºC for 5 min. To construct the sequencing libraries, the resulting mixture underwent 13-14 cycles of PCR amplification, ensuring an adequate amount of library material for sequencing purposes. The libraries were size-selected using VAHTS DNA Clean Beads (Vazyme) and sequenced on a Novaseq 6000 (Novogene) with paired-end reads of 150 bp.