Dynabeads protein a g magnetic beads

Chromatin immunoprecipitation analysis was performed as described previously (Jiang et al., 2018b). In brief, ESCC cells KYSE150, KYSE510, and TE3 were treated with THZ1 (100 nm, 12 h), and then, 1 × 10⁷ cells were cross‐linked with 1% formaldehyde solution (Thermo Fisher Scientific) and neutralized by 1.25 m glycine. Cross‐linked cells were lysed and sonicated (Covaris E220, Woburn, MA, USA) to release 100–00 bp fragments. Anti‐KLF5 (Santa Cruz Biotechnology; sc‐398470x), anti‐TCF3 (Santa Cruz Biotechnology; sc‐166411x), or normal IgG was added to each sonicated chromatin and incubated at 4 °C overnight. Then, these complexes were conjugated to Dynabeads protein A/G magnetic beads (Invitrogen) for 4 h at 4 °C. After incubation, DNA was eluted from immunoprecipitate complexes, reverse cross‐linked, and purified with QIAquick PCR purification kit (QIAGEN, Hilden, Germany).
The purified DNA was analyzed by real‐time PCR with the use of LINC00094 super‐enhancer‐specific primers. Primers for ChIP‐PCR were shown in Table S5. Relative enrichment was normalized to input. IgG antibody was used as a negative control.

ChIP-PCR assays were performed as previously described [61 (link)]. In brief, 1 × 10⁷ KYSE30 and KYSE150 cells were crosslinked with formaldehyde (1%) (28908, Thermo Fisher) and neutralized with glycine. Cells were lysed, and DNA was disrupted by sonication (Covaris E220, Woburn, MA, USA). Each ultrasonic product was divided into two equal volumes. Anti-ELK1 and normal IgG were added to each volume and incubated at 4 °C overnight. Dynabeads protein A/G magnetic beads (Invitrogen) were added for 4 h at 4 °C. Complexes were immunoprecipitated, and DNA was eluted and purified using QIAquick PCR Purification Kit (28106, QIAGEN, Germany). The immunoprecipitated DNA was quantified via PCR with SOCS3 promoter-specific primers and separated on a 1.8% agarose gel. The primer sequences used for ChIP-PCR are listed in Supplementary Table S5. The relative enrichment was normalized to a 1% input. IgG antibody was used as a negative control.