title: Illumina HiSeq 2500 paired end sequencing; RNA-seq comparison of immortalised myoblasts from dystrophic and WT mice
design: RNA-seq comparison of immortalised myoblasts from dystrophic and WT mice
library_construction_protocol: Immortalised myoblast cell lines were cultured in immorto conditions to produce 4 populations of each genotype growing apart for 2 passages and then replated and cultured for 48 hours in primary myoblast conditions at 37°C with 5 % CO2 in IFN-γ free medium. Immortalized mdx mouse and dystrophin-positive control (w/t) myoblasts were used. These two cell lines were of the same genetic background, as the two mouse strains from which the cells were derived originated from the same parental inbred mouse strain H2Kb- tsA58 (“immorto mouse”), expressing the SV40 large T antigen gene with the tsA58 mutation. The H2Kb-tsA58 mdx strain was established by crossing male mouse homozygous for H2Kb-tsA58 with female mdx mice. Mdx and w/t cells used here are highly homogenous populations differing only in the presence or absence of a point mutation in the dystrophin gene exhibiting muscle phenotype and myogenic potential following differentiation. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum, 4 mM L-glutamine, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 20 unit/ml murine γ-interferon (Invitrogen, San Diego, CA, USA) at 32°C. Under such conditions a differentiation of these cells was efficiently prevented. Moreover both cell lines were also cultured at 37°C in absence of γ-interferon in KnockOut DMEM (Invitrogen) supplemented with 10% v/v KSR (Knockout Serum Replacement, Invitrogen), 5% v/v DHS (Donor Horse Serum, Sera Labs) and 2 mM L-glutamine. Under these conditions the cells exhibits phenotypic properties of primary myoblasts. Total RNA was extracted using an RNEasy plus universal mini kit (QIAGEN 73404) following manufacturer's instructions. Briefly, cells were washed 3 times with warm DPBS before being lysed directly on the culture plate by adding QIAZOL lysing buffer and using a cell scraper to disrupt the cells. Samples were then treated as per provided protocol. Libraries were prepared using an Illumina TruSeq stranded total RNA kit, including ribodepletion using the Ribo-Zero Human/Mouse/Rat kit to remove ribosomal RNA.
attributes.tag: Experimental Factor: disease
attributes.value: Duchenne muscular dystrophy