Genome-wide Kdm1a binding in X chromosome by high throughput sequencing [ChIP-Seq]

We analyzed KDM1A (LSD1) occupancy in the Xi during somatic cell reprogramming of female mouse cells. We use MEFs from hybrid embryos by crossing male Mus spretus and female Mus musculus domesticus C57BL/6J to distiguish genome DNA from the Xi. We found a possible physical and/or functional regulation of KDM1A during the X chromosome reactivation in the intiation site on the Xi. Overall design: Chromatins were isolated from MEF, and cells reprogrammed by SeVdp(fKOSMaB) with or without 30 nM Shield1 at day 20. ChIP was performed using anti-LSD1 antibody (ab17721, abcam). Libraries were sequenced on an Illumina NovaSeq 6000 and data was analyzed allele-specifically by variantcall using bcftools mpileup.

本研究分析了雌性小鼠细胞体细胞重编程过程中，失活X染色体（Xi，X-inactive chromosome）上赖氨酸特异性去甲基化酶1（KDM1A, LSD1）的结合富集情况。本研究通过将雄性西班牙小家鼠（Mus spretus）与雌性驯化小家鼠C57BL/6J（Mus musculus domesticus C57BL/6J）杂交获得杂交胚胎，分离得到小鼠胚胎成纤维细胞（MEFs，Mouse Embryo Fibroblasts），以此区分来自失活X染色体的基因组DNA。本研究发现，在失活X染色体的起始位点处，KDM1A可能在X染色体重激活过程中发挥物理层面或功能层面的调控作用。整体实验设计：从初始小鼠胚胎成纤维细胞以及重编程第20天的细胞中分离染色质；重编程过程采用SeVdp(fKOSMaB)载体，并设置添加30 nM Shield1与不添加Shield1两组对照。采用抗LSD1抗体（ab17721，abcam公司）进行染色质免疫共沉淀（ChIP，Chromatin Immunoprecipitation）实验。构建的测序文库在Illumina NovaSeq 6000测序平台上完成测序，随后通过bcftools mpileup进行变异检测，实现等位基因特异性的数据分析。