Chd4 ensures stem cell lineage fidelity during skeletal muscle regeneration. 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-α7-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–/α7-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).

染色质重塑因子Chd4是核小体重塑与去乙酰化（NuRD）抑制复合物的成员，对卫星细胞的扩增及再生功能至关重要。实验整体设计：使用手术刀解剖前肢与后肢肌肉，将组织置于预冷的DMEM（杜尔贝科改良伊格尔培养基，Dulbecco's Modified Eagle Medium，Gibco）中回收。将肌肉组织充分剪碎后，使用含5 mg/mL Liberase（罗氏/西格玛奥德里奇）、0.03% Dispase II（西格玛）、DMEM、1%青霉素/链霉素、0.2%牛血清白蛋白（BSA，西格玛）、1 M氯化钙及1 M氯化镁的消化体系，于37℃下进行组织消化。样本于4℃下以50g离心10分钟。上清液先后通过100 μm及70 μm细胞筛过滤，随后以1700 rpm离心15分钟。沉淀重悬于1×裂解缓冲液（BD）中，孵育10分钟。将样本重新重悬于预冷DMEM中，经40 μm细胞筛过滤后，再次以1700 rpm离心15分钟。将细胞重悬于磷酸盐缓冲液（PBS）中，加入2.5%山羊血清（流式缓冲液）。样本于4℃下与以下抗体孵育30分钟：抗CD45 PE-Cy7（Biolegend）、抗Sca1 PE-Cy7（Biolegend）、抗α7整合素PE（Ablab.ca）及抗CD34 Alexa-647（BD Pharmingen）。样本以1700 rpm离心15分钟后，最终重悬于含1 μg/mL DAPI的流式缓冲液中。抗体标记完成后，通过BD Influx流式细胞分选仪进行荧光激活细胞分选（FACS），分选出SCA1–/CD45–/α7整合素+/CD34+细胞，随后开展RNA测序（RNA-seq）与ATAC测序（ATAC-seq）。样本与文件名称中已标注分组（体外Invitro与体内Invivo）。