GSM9376465: MRC_seq_siREXO4_2_Rep2; Homo sapiens; OTHER
The S9.6 CUT&Tag assay was first performed at a larger scale. Briefly, a total of 2 × 10⁶ cells were washed twice with 1 ml of wash buffer, resuspended in 1 ml of the same buffer, and bound to 200 l of pre-activated ConA beads. After washing, the bead-bound cells were resuspended in 1 mL of antibody buffer and incubated with 20 g of S9.6 primary antibody overnight at 4 °C. Following secondary antibody incubation and washes in dig-wash buffer, cells were resuspended in 2 ml of 0.2 M pAG-Tn5 complex in dig-300 buffer. Tagmentation was carried out in 1 mL of tagmentation buffer at 37 °C for 1 hr. The reaction was stopped with 40 l of 10% SDS and incubated at 55 °C for 15 min. DNA was enriched and purified using DNA Extract Beads Pro (Vazyme) followed by the strand displacement reaction. To prepare for m6A immunoprecipitation, the DNA-bound beads were washed three times with 1 × NEBNext dA-Tailing Reaction Buffer (NEB), resuspended in 5 U/l Klenow fragment (3'→5' exo-) (NEB) in 1 × NEBNext dA-Tailing Reaction Buffer (NEB), and incubated at 37 °C for 30 min. After washing three times with 1 × Quick Ligase Buffer (NEB), the DNA-bound beads were mixed with 2 l Quick Ligase (NEB) and 2 l hairpin-like MRC-seq adapter (0.5 M) in 1 × Quick Ligase Buffer (NEB) and incubated at 25 °C for 20 min. The ligation reaction mix was then removed, and the DNA-bound beads were resuspended in 20 l 1 × CUT&Tag Stop Buffer (Vazyme) at 95 °C for 20 min. After heat denaturation and quenching on ice, the supernatant containing circularized and single-stranded RNA-DNA was collected and then mixed with the m6A antibody-protein G beads complex in 1 mL of m6A immunoprecipitation buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40, and 1 × protease inhibitor) for 2 hr at 4 °C. Following four washes with m6A immunoprecipitation buffer, bead-bound nucleic acids were digested with the USER enzyme (NEB). For library construction, the resulting mixture was used directly for library construction, which included PCR amplification (15-16 cycles) and purification with VAHTS DNA Clean Beads (Vazyme). Finally, sequencing was performed on an Illumina NovaSeq 6000 platform (Novogene) with 150-bp paired-end reads.