RNA-seq from myogenic satellite cells from a translational porcine model for Duchenne muscular dystrophy, heterozygous female carriers, and their healthy wild-type counterpart
RNA-seq from myogenic satellite cells from a translational porcine model for Duchenne muscular dystrophy, heterozygous female carriers, and their healthy wild-type counterpart
A detailed protocol for the isolation of myogenic satellite cells (SCs) for sample collection is provided in Franzmeier et al., 2025. In summary, tissue biopsies from intercostalis and pectoralis muscles were collected from WT, DMD, and HET animals immediately after euthanasia, and were minced manually with a scalpel blade. After enzymatic digestion with protease (Streptomyces griseus, Sigma Aldrich, 3.5 U/mg) and collagenase (Clostridium histolyticum, Sigma Aldrich, 800 U/mg), cell suspensions were subjected to magnetically activated cell sorting (MACS) using an indirect labelling approach and two sequential separation steps. Unwanted endothelial and hematopoietic cells were depleted by adding rabbit anti-CD31 (1:200, ab28364, Abcam) and rabbit anti-CD45 (1:200, ab10558, Abcam) antibodies together with magnetically conjugated anti-rabbit IgG(-) secondary antibodies (1:15, Miltenyi Biotec) to the cell suspension. Labelled cells were retained within a magnetic column placed on the MACS separator, and the flow-through was used for the subsequent separation step. SCs were targeted with mouse anti-Integrin-1 (1:200, abcam30388, Abcam), mouse anti-NCAM1 (1:200, ab9018, Abcam), and mouse anti-M-cadherin (1:200, sc-374093, Santa Cruz) and magnetically conjugated secondary anti-mouse IgG(-) antibodies (1:15, Miltenyi Biotec); again labelled cells were retained in the column allowing enrichment of the desired SC population. Purified SC were cultivated under standard conditions in Ham's F10 Medium (GIBCO, Thermo Fisher) supplemented with 20% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin (GIBCO), and 10 μg recombinant human basic fibroblast growth factor (PEPro Tech). Myoblast differentiation was induced by nutrient starvation using medium with 5% horse serum (Biozol). Total RNA from WT, DMD, and HET SCs was isolated using the NEB Monarch RNA Miniprep Kit in accordance with the manufacturer's instructions. RNA concentration was determined with a Nanodrop 2000c spectrophotometer (Thermo Scientific) before proceeding with subsequent experiments. Cells were harvested at p6 and p7 after cultivation in normal growth medium to recapture the proliferation stage (PROL), and after 10 days in medium with reduced serum amount for the differentiation stage (DIFF). Gene expression was quantified using an adapted 3'm RNA-seq protocol. 25 ng of total RNA was used for each sample and analysed as a technical duplicate. For library construction, polyA+ RNAs were selected with a unique molecular identifier (UMI) and a well-barcode for early pooling directly after cDNA synthesis. By RT-PCR using a template switch oligo with an integrated Illumina adapter sequence, second-strand synthesis was performed. Subsequently, purification and exonuclease◦I treatment were executed, followed by library pool amplification using NexteraXT DNA library prep Kit (Illumina). DNA was tagmented with Nextera transposons, and for library amplification, a 3' enrichment PCR was performed during which i7 and i5 adapters were attached. After purification, the quantity of the library pool was measured with a Qubit Fluormeter 4.0 (Thermo Fisher Scientific) as well as average fragment size with a Labchip GX Touch 24 (Perkin Elmer/Revvity). Please see also Franzmeier et al., 2025.