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. Overall design: 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)