Ercc rna spike in control

HepG2 cells with stably expressed shRNAs against IGF2BPs or shNS were seeded into 6-well plates to get 50% confluency after 24 hours. Cells were treated with 5 μg/ml actinomycin D and collected at indicated time points. The total RNA was extracted by miRNeasy Kit (Qiagen) and analyzed by RT-PCR and RNA-seq. For RNA sequencing, equal amount of ERCC RNA spike-in control (Thermo Fisher Scientific) was added to the total RNA samples as internal controls before library construction. Sequencing libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit. RNA stability profiling was generated from two biological replicates.
The turnover rate and half-life of mRNA was estimated according to previously published paper⁴⁴ . Since actinomycin D treatment results in transcription stalling, the change of mRNA concentration at a given time (dC/dt) is proportional to the constant of mRNA decay (K_decay) and mRNA concentration (C), leading to following equation:
$$\text{dC}/\text{dt}=-{\mathrm{k}}_{\text{decay}}\mathrm{C}$$
Thus the mRNA degradation rate K_decay was estimated by:
$$\text{Ln}(\mathrm{C}/{\mathrm{C}}_0)=-{\mathrm{k}}_{\text{decay}}\mathrm{t}$$
To calculate the mRNA half-life (t_1/2), when 50% of mRNA is decayed (ie. C/C₀=1/2), the equation was:
$$\text{Ln}(1/2)=-{\mathrm{k}}_{\text{decay}}{\mathrm{t}}_{1/2}$$
From where:
$${\mathrm{t}}_{1/2}=\text{ln}2/{\mathrm{k}}_{\text{decay}}$$