Anlotinib 1
Homo sapiens
Protocols: Cells were maintained in RPMI 1640 medium (Gibco, USA) supplementary with 10% fetal bovin serum (FBS) (Gibco, USA) and 100 units/mL of penicillin and streptomycin (Gibco, USA). Cells were incubated at 37℃ in a humidified atmosphere of 5% CO2 NCI-H1975 cells were cultured in 6-well plates for 24 hours, and then were treated with Anlotinib (8 μg/mL) for 24 hours. Trizol reagent (Life Technologies, Inc., USA) was used to lyse cell samples, followed by total RNA isolation using standard procedures. mRNA was isolated using Oligotex mRNA Mini Kit (Qiagen, Germany). Residual genomic DNAs were degraded by adding DNase (NEB, USA), incubated for 30 minutes at 37℃. 100 ng mRNA from each sample was reverse-transcribed into cDNA and fragmented to 100-300 bp length. NEBNext Ultra Directional RNA Library Prep Kit (NEB, USA) was used to end-repair and adapter-ligate the fragment cDNA. Samples were amplified 10-12 cycles in a thermal cycler, in the medium of PCR Master Mix supplement with Q5 High-Fidelity DNA Polymerase (NEB, USA).