NOMe-Seq analysis of fixed budding yeast nuclei with two DNA methyltransferases

NOMe-Seq analysis was performed with two cytosine C-5 DNA methyltransferases recognizing GC and CC dinucleotides Overall design: Spheroplasts were prepared with treating S288C cells with Zymolyase 100T. Then, 1% formaldehyde was added to fix the cells. After washing the cells with 1 M sorbitol, the yeast nuclei were prepared with a solution containing 0.1% NP-40. The nuclei were suspended in methylation buffer, and cytosine C-5 DNA methyltransferase was added. The solution was incubated at 37°C for 1 hr and collected nuclei. Then genomic DNA was extracted, and library preparation was performed with tPBAT protocol (Miura F et al., NAR, 2019)NOMe-Seq 分析采用了两种可识别GC与CC二核苷酸的胞嘧啶C-5 DNA甲基转移酶。整体实验设计如下：使用Zymolyase 100T处理S288C细胞以制备原生质体；随后加入1%甲醛固定细胞。经1 M山梨醇洗涤细胞后，采用含0.1% NP-40的溶液制备酵母细胞核。将细胞核重悬于甲基化缓冲液中并加入胞嘧啶C-5 DNA甲基转移酶，于37℃孵育1小时后收集细胞核。随后提取基因组DNA，并通过tPBAT协议（Miura F等，《核酸研究》，2019年）完成文库构建。