Time-course analysis of gene expression during mouse embryonic stem cell differentiation into cardiomyocytes: impact of Npl deficiency.
Time-course analysis of gene expression during mouse embryonic stem cell differentiation into cardiomyocytes: impact of Npl deficiency.
ells were harvested at designated time points (D0, D2, D4, and D14). The culture medium was aspirated and discarded. Cell layers were gently washed once with ice-cold DPBS to remove residual serum and media components. Cells were then collected by mechanical scraping using a cell scraper in the presence of DPBS or lysis buffer. Total RNA was extracted from cells using QIAzol Lysis Reagent (Qiagen) followed by purification with the Reasy Mini Kit (Qiagen) according to the manufacturerâ€™s instructions. RNA concentration and purity were assessed using a Nanodrop 2000 spectrophotometer (Thermo Scientific), with samples exhibiting A260/A280 ratios between 1.8 and 2.1 and A260/A230 ratios > 2.0 considered suitable for downstream analysis. RNA integrity was further verified using an Agilent 5400 Fragment Analyzer, with RNA integrity numbers (RIN) > 8.0 required for library preparation. RNA samples were sent to Novogene (UK) for library construction, quality control and sequencing.