Expression profiling by high throughput sequencing
Summary
RNA sequencing was performed on proliferating and differentiating emerin-null myogenic progenitors expressing emerin and EDMD-causing emerin mutants to identify molecular pathways implicated in Emery-Dreifuss Muscular Dystrophy.
Overall design
Wildtype and EMD-/y H2K myogenic progenitors were seeded in 6-well dishes coated with 0.01% gelatin at a density of 232,000 cells/well for isolation of total RNA during differentiation. 297,000 cells /well of vector control and emerin mutant cell lines were plated in 6-well dishes coated with 0.01% gelatin. Cells were incubated overnight in proliferative media. In the morning, cell lines were switched to differentiation media. RNA was isolated after 0 hours (to account for changes due to cell density), 24 hours, 48 hours, and 72 hours of differentiation. RNA was isolated using the mRNeasy Plus Kit (Qiagen, Germantown, MD. USA, #74136) per manufacturer’s instructions. Three technical replicates per sample were performed for each biological replicate (n=2).
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