Chd4 ensures stem cell lineage fidelity during skeletal muscle regeneration

The chromatin remodeler Chd4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. Overall design: Dissection of fore and hind limb muscles was done with a scalpel and retrieved in cold DMEM (Dulbecco's Modified Eagle Medium, Gibco). Muscle tissues were finely minced and digested with Liberase 5 mg/mL (Roche/Sigma Aldrich), 0.03% Dispase II (Sigma), DMEM, 1% penicillin/streptomycin, BSA 0.2% (Sigma), 1 M CaCl2, and 1 M MgCl2 at 37ºC. Samples were then centrifuged 10 min at 50g at 4ºC. Supernatant was filtered through 100-µm and then a 70-µm cell strainers filters and then centrifuged at 1700 rpm for 15 min. Pellets were then resuspended in Lysis Buffer 1? (BD) and incubated for 10 min. Samples were resuspended in cold DMEM, filtered through 40-µm cell strainer filters, and centrifuged again for 15 min at 1700 rpm. Cells were resuspended in PBS, and 2.5% of goat serum (FACS buffer). Samples were incubated 30 min at 4ºC with the following antibodies: anti-CD45 PE-Cy7 (Biolegend), anti-Sca1 PE-Cy7 (Biolegend), anti-a7-integrin PE (Ablab.ca), and anti-CD34 Alexa-647 (BDPharmingen). Samples were centrifuged at 1700 rpm for 15 min and finally resuspended in FACS buffer with 1 µg/mL DAPI. After antibody labelling, SCA1–/CD45–/a7-integrin+/CD34+ cells were sorted by fluorescence-activated cell sorting (FACS) in the BD Influx cell sorter, and processed for RNA-seq and ATAC-seq. Sets are indicated in the sample and file names (Invitro and Invivo).