Genome-wide Lsd1 chromatin occupancy in myoblast C2C12 cells by chromatin immunoprecipitation using an Lsd1 antibody followed by massive parallel sequencing

Purpose: The aim of this study is to identify the Lsd1 genome binding profile in myoblast C2C12 cells during myogenic and adipogenic differentiation. ChIP-seq libraries were prepared, sequenced using the standard Illumina protocol (HiSeq2000, single read, 50 bp v3), and mapped to the mouse mm10 reference genome by Bowtie. Data were further analyzed using the peak finding algorithm MACS 1.4.2. Homer software was used to annotate peaks, and all peaks with false discovery rate less than 1 % were included. For ChIP-seq experiment, C2C12 cells were fixed for 6 minutes with 1 % PFA at 4 °C followed by 5 minutes blocking in 125 mM glycine. Nuclei preparation was performed using previously described NEXON protocol (Arrigoni et al., 2015), by sonicating the cells with Covaris E220 focused ultrasonicator for 2 minutes at peak power 75 W, duty factor 2 % and 200 Cycles/burst at 4 °C. Chromatin was sheared using Covaris E220 focused ultrasonicator to obtain a fragment size distribution of 100–800 bp (peak power: 140 W; Duty factor: 5 %; Cycles/burst: 200, water temperature 4 °C). Chromatin was immunopercipitated using homemade antibody directed against C-terminus of Lsd1 protein (Biogenes, 20752).