ChIP-seq of mus musculus
Briefly, 50 g of E15.5 brain tissue was washed with cold PBS and cross-linked with 1% formaldehyde, then quenched with glycine. The tissue was lysed in buffer containing Tris-HCl, EDTA, SDS, and protease inhibitors, and chromatin was isolated and sonicated to 200500 bp fragments. A portion of chromatin was saved as input, and the rest was incubated overnight with 10 g of anti-Zic3 antibody at 4 C. The next day, protein G beads were added and incubated for 3 h, followed by a series of washes. DNA was eluted, treated with RNase A and proteinase K, and purified. Libraries were prepared using the NEXTFLEX ChIP-Seq Kit (Bioo Scientific) and sequenced on an Illumina NovaSeq 6000 using 150 bp paired-end reads. For ChIP-Qpcr, Immunoprecipitated DNA was analyzed by quantitative PCR using specific primers. Reactions were performed with ChamQ SYBR Color qPCR Master Mix. Enrichment was normalized to input DNA and calculated using the 2^(-Ct) method