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The ability of SPI1 to bind to ST8SIA1 was detected by a ChIP assay. The cells were fixed with 16% methanol and cross-linked, then treated with lysis buffer and sonicated. The cells were subsequently incubated with an anti-SPI1 antibody (1: 1000, sc-365,208, Santa Cruz Biotechnology Inc.) overnight. Next, beads were added to harvest the protein-DNA complex. Then decrosslinking was performed with addition of 5 mmol/l NaCl to retrieve DNA. The enrichment of SPI1was examined using RT-qPCR.

The ability of NR2F1-AS1 to bind SPI1 was detected using the RIP kit (Millipore Sigma, Burlington, MA, USA). The cells were lysed with an equal volume of RIPA lysis (P0013B, Beyotime Biotechnology) in an ice bath for 5 min. Part of the cell extract was saved as the input, and the remaining was incubated with anti-IgG (1: 100, ab109489, Abcam) or anti-SPI1 (1: 1000, sc-365,208, Santa Cruz Biotechnology Inc.) antibodies for co-precipitation. The bead-protein complex was collected on a magnetic base. RNA was extracted from the co-precipitated samples and Input samples after detachment from the bead using protease K. The expression of NR2F1-AS1 was determined using RT-qPCR.