ChIP assays were performed according to the manufacturer’s instructions with modifications using the ChIP-IT kit (Active Motif, USA). Briefly, the TH17 cells were fixed with 1% formaldehyde, and then, the cross-linked chromatin was sonicated in a 4°C water bath using a Bioruptor Pico sonicator (Diagenode) to obtain DNA fragments between 150 and 500 base pairs (bp) in size. For Cxxc1 ChIP-seq, 5 × 106 TH17 of cells and 6 μg of Cxxc1 antibody were used for each sample. For H3K4me3 ChIP-seq, 3 × 106 of TH17 cells and 4 μg of H3K4me3 antibody were used for each sample.

The immunoprecipitated DNA was purified and subjected to sequencing library preparation using a VAHTSTM Universal DNA Library Prep Kit for Illumina V2 (Vazyme Biotech Co. Ltd.) according to the manufacturer’s protocol. The DNA libraries were then sequenced with an Illumina HiSeq X Ten system at Veritas Genetics in Hangzhou.

Sequenced reads of 150 bp were obtained using the CASAVA 1.8.2 package (Illumina). All reads were mapped to the mm10 mouse genome, and uniquely mapped reads were subjected to a further peak identification process. MACS2_V2.1.1 was used to identify significant peaks (q = 0.05) with both input DNA and ChIP DNA in Cxxc1-deficient cells as controls. The output of the peak files was converted by IGV browser. To calculate the tag density for Cxxc1-binding sites or H3K4me3 modifications around the TSS or at the centers of CGIs, uniquely mapped tags were summarized in 100-bp windows, and all window tag counts were normalized by the total number of bases in the windows and the total read number for the given sample.

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