RNA-seq of human colorectal cancer HCT116 and HT29 cells by the distinct conditions of trypsin digestion
RNA-seq of human colorectal cancer HCT116 and HT29 cells by the distinct conditions of trypsin digestion
For cell digestion, medium was removed, and cells with 80% density were simply washed by PBS twice. 1 mL 0.25% trypsin-EDTA (Thermo Fisher Scientific) was added into 10 cm dish, and uniformly dispersed for 0.5 min at 37 oC, then quenched immediately by 4 mL regular medium. The suspended cells were harvested after centrifugation of 5 mL supernatant. 1 mL trypsin was added again to digest the rest of cells for 2 min at 37 oC, then harvested the suspended cells as same procedure as above. The third round of 1 mL trypsin was added to digest the rest of cells for 4 min at 37 oC. These three batches of cells were used for the consequent experiments. polysome-associated RNA was extracted from at least 5 × 106 cells using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China) according to the manufacturer's protocol.  Isolation of mRNA was performed using the NEBNext PolyA mRNA Magnetic Isolation Module (New England Biolabs, USA) and the mRNA was further used with the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) for sequencing library preparation. The libraries were quantified with a Qubit 4.0 (Thermo Fisher Scientific), and the distribution of fragment sizes was determined using an Agilent Bioanalyzer 2100 (Agilent, USA).