Protocols: Gastrocnemii from 8-week-old male mice were used for myoblast extraction and culture. Gastrocnemius was chosen as it is considered a fast muscle with predominantly IIB fibres (Augusto et al., 2004) and therefore more prone to dystrophic changes and producing a relatively homogenous population of myoblasts. Briefly, muscles were dissected free of fat and connective tissue before enzymatic digestion by 0.2 % type IV collagenase (Sigma, C5138) in DMEM (Gibco 31885-023) for 90 min at 37°C, 5 % CO2. The digested muscles were then rinsed in plating medium (10 % horse serum, Gibco 26050-088; 0.5 % chicken embryo extract, Seralab CE-650-J; penicillin / streptomycin, Sigma P0781; in DMEM, Gibco 31885-023) incubated at 37°C, 5 % CO2. Digested muscles were then disrupted by successive passages in 50 mL, 25 mL and 10 mL serological pipettes. The freed muscle fibres were transferred to a dish containing plating medium. The dish was then placed under a stereo microscope and 200 live fibres were transferred to another dish containing plating medium. This procedure was repeated 2 more times until 150 fibres were transferred to a third dish. 120 live fibres were then transferred to a tube containing proliferating medium (20 % foetal bovine serum, Gibco 10500-064; 10 % horse serum, Gibco 26050-088; 0.5 % chicken embryo extract, Seralab CE-650-J; penicillin / streptomycin, Sigma P0781; in DMEM, Gibco 31885-023). The resulting suspension of muscle fibres in medium was then disrupted by passing it through an 18-gauge needle 10 times before passing the resulting solution through a 40 μm cell strainer (BD Falcon 352340). The resulting cell suspension was transferred to a collagen I (Sigma C8919) coated cell culture dish and proliferating medium was added to obtain an appropriate volume for the dish. The cells were then cultured and expanded as needed for assays and experiments. Cells were lifted from dishes using Accutase (Biowest L0950). Myoblasts isolated from gastrocnemii from 3 individual mice per experimental group were treated as biological replicates. Total RNA was extracted from Dmdmdx, Dmdmdx-βgeo and corresponding wild-type myoblasts using RNEasy Plus Universal mini kits (Qiagen 73404). Briefly, cells were washed 2 times with warm DPBS (Gibco 14190144) before being lysed by applying QIAzol directly to the freshly washed cells. Lysate was then homogenised by passing it through a 25-gauge needle 20 times. Samples were then processed according to kit manufacturer's instructions. RNA quality and concentration were measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). RNA integrity was assessed using electrophoresis of 100 ng of total RNA in a 1 % agarose gel (Sigma A4718) in TAE buffer or using an automated capillary electrophoresis system (2100 Bioanalyzer Instrument G2939BA, Agilent) using a kit assay (Agilent RNA 6000 Nano Kit 5067-1511, Agilent). Total RNA samples (N=3 per group) were processed by TeragenEtex (Republic of Korea) using an Agilent Bioanalyzer 2100 for quality assessment of samples with a threshold of 7.0 for the RNA integrity number (RIN). An Illumina TruSeq stranded total RNA kit was used to generate libraries following a ribodepletion step using a Ribo-Zero Human/Mouse/Rat kit.
ENA-FIRST-PUBLIC
2022-09-14
ENA-LAST-UPDATE
2022-09-14
External Id
SAMEA8521104
INSDC center alias
University of Portsmouth
INSDC center name
University of Portsmouth
INSDC first public
2022-09-14T20:17:10Z
INSDC last update
2022-09-14T20:17:10Z
INSDC status
public
Submitter Id
E-MTAB-10322:WT2
age
8
broker name
ArrayExpress
cell_type
myoblast
common name
house mouse
developmental stage
adult
disease
normal
genotype
wild type genotype
organism part
gastrocnemius
sample name
E-MTAB-10322:WT2
sex
male
strain
C57BL/6J x C57BL/10