The role of Lyar in the regulation of cell cycle checkpoints
Cells were plated on 10 cm plates and grown for 48 hours. Subsequently, the cells were pelleted, washed twice with PBS, and stored at -80Â°C. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions.The concentration of isolated RNA was measured by NanoDrop. This step was performed by Novogene Europe. Messenger RNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. After fragmentation, the first strand cDNA was synthesizedusing random hexamer primers, followed by the second strand cDNAsynthesis using either dUTP for directional library or dTTP for non-directional library.For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantificationand bioanalyzer for size distribution detection. Quantified libraries were and sequenced on Illumina platforms, according to effective library concentration and data amount.