MBD2 and H3K4Me3 CUT&RUN-sequencing in C2C12 cells undergoing myogenic differentiation. MBD2 and H3K4Me3 CUT&RUN-sequencing in C2C12 cells undergoing myogenic differentiation

MBD2 genome-binding landscape was assessed by CUT&RUN-sequencing in differentiating C2C12 cells. H3K4Me3 CUT&RUN-sequencing was performed as a positive control. Negative control experiment was also performed using a rabbit isotype control monoclonal IgG. Overall design: C2C12 cells that have undergone myogenic differentiation were harvested at 0, 1, and 5 days for CUT&RUN-sequencing. C2C12 myotubes and reserve cells were separated by mild trypsinization. CUT&RUN-sequencing library was prepared using a Cell Signaling Technology CUT&RUN assay kit. Sequencing libraries were prepared using NEBNext Ultra II DNA library prep kit for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7645 and E7600). Libraries were quantified and analyzed using Qubit dsDNA HS assay kit (Thermo Fisher Scientific) and a TapeStation 2200 with high-sensitivity DNA kit (Agilent). Libraries were sequenced in paired-end 150 base pair mode on the Illumina NovaSeq 6000 platform. Paired-end fragments were trimmed, mapped to the mm10 genome, and filtered using Trim Galore, Bowtie2 , and SAMtools , respectively. Peaks were identified using MACS2 and further filtered and analyzed with in-house Python scripts that leveraged pyBigWig and pyBedTools packages. GO term analyses (biological processes) were performed on http://metascape.org. Genomic annotation of the peaks was conducted using ChIPseeker. CUT&RUN-sequencing data were plotted using karyoploteR.

本研究采用CUT&RUN测序（CUT&RUN-sequencing）对分化过程中小鼠成肌细胞系C2C12的甲基化CpG结合域蛋白2（MBD2）全基因组结合图谱进行了检测。以组蛋白H3赖氨酸4三甲基化（H3K4Me3）的CUT&RUN测序作为阳性对照，同时采用兔同源型对照单克隆IgG开展了阴性对照实验。整体实验设计：对已完成肌源性分化的C2C12细胞，分别于分化第0、1、5天收集样本用于CUT&RUN测序。通过温和胰酶消化法分离C2C12肌管与储备细胞。CUT&RUN测序文库的制备采用Cell Signaling Technology品牌的CUT&RUN检测试剂盒。测序文库构建同时使用了适配因美纳（Illumina）平台的NEBNext Ultra II DNA文库制备试剂盒，以及适配Illumina平台的NEBNext多重寡核苷酸试剂盒（纽英伦生物科技，New England Biolabs，货号E7645与E7600）。文库的定量与质量分析分别采用Qubit dsDNA HS检测试剂盒（赛默飞世尔科技，Thermo Fisher Scientific）以及搭载高灵敏度DNA试剂盒的TapeStation 2200系统（安捷伦，Agilent）。文库在因美纳（Illumina）NovaSeq 6000测序平台上以150bp双端测序模式完成测序。双端测序片段依次通过Trim Galore、Bowtie2与SAMtools进行序列修剪、小鼠参考基因组mm10比对与过滤质控。采用MACS2软件识别基因组结合峰，并借助自研Python脚本结合pyBigWig与pyBedTools工具包对结合峰开展后续过滤与分析。在metascape.org平台上开展GO基因本体分析（生物学过程分支）。结合峰的基因组注释采用ChIPseeker软件完成。CUT&RUN测序数据的可视化采用karyoploteR软件完成。