4C-seq experiment on mouse skeletal muscle and DP thymocytes

Purpose: The aim of this study is to investigate enhancer promoter interactions in skeletal muscle Methods: 50 ng of purified DNA were used as a template for PCR with bait-specific primers (Table S5) containing Illumina adapter termini. PCR reactions were pooled and, after primer removal with 1.8x AMPure XP beads, DNA was sequenced with the HiSeq 4000 (Illumina), as single-end 50 bp reads. Sequences were trimmed to remove primer and bait fragment sequence with the sabre tool (https://github.com/najoshi/sabre), mapped to the genome with Bowtie and converted to restriction fragment space as in van de Werken et al., 2012 (van de Werken et al., 2012). Interactions were called on single 4C experiments with peakC (Geeven et al., 2018), using a sliding window of 21 fragments. Nuclei were extracted from skeletal muscles from 10-week-old mice as described in Joshi 2017. Alternatively, 4 week old thymus was dissected, cells were washed and filtered in PBS, and then sorted by FACS with anti-CD4-PE and anti-CD8a-FITC antibodies (eBioScience)

研究目的：本研究旨在探究骨骼肌中增强子与启动子的相互作用。
研究方法：取50 ng纯化后的DNA作为模板，使用带有Illumina接头末端的诱饵特异性引物（补充表S5）进行PCR扩增。将所有PCR反应体系混匀后，采用1.8倍体积的AMPure XP磁珠去除引物，随后使用Illumina HiSeq 4000平台进行单端50 bp读长测序。使用sabre工具（https://github.com/najoshi/sabre）对测序序列进行修剪，以去除引物与诱饵片段序列；通过Bowtie将序列比对至参考基因组，并按照van de Werken等人2012年的研究方法（van de Werken et al., 2012）将其转换为限制性酶切片段空间。利用peakC工具（Geeven等人，2018），以21个片段的滑动窗口对单次4C实验进行相互作用位点识别。按照Joshi 2017年的方法，从10周龄小鼠的骨骼肌中提取细胞核。此外，也可取4周龄小鼠的胸腺进行解剖，将细胞置于磷酸盐缓冲液（PBS）中洗涤、过滤后，使用抗CD4-PE与抗CD8a-FITC抗体（eBioScience）通过荧光激活细胞分选术（FACS）进行细胞分选。