title: GSM8443016: HCT16T61AKO2 Ribo-Seq; Homo sapiens; OTHER
library_construction_protocol: Cycloheximide (Sigma, St Louis, MO, USA) was added to cell cultures medium to a final concentration of 100 μg/ml and incubated for 2 min. The clean cell pellet was lysed in 1mL lysis buffer at 4℃ for 10 min followed by incubation of 10μL RNase A (BBI, B600476) at room temperature for 30 min. The RNAs were extracted by Trizol and rRNA was removed with SP-ribo-Pools (Forever Star, FS-R1056-04) followed by clean-up with VAHTS RNA Clean Beads (Vazyme, N412).  The clean RNA samples were then treated with T4 polynucleotide Kinase (PNK, Vazyme, N102) to create RNA fragments with 3'-hydroxyl and a 5'-phosphate that are compatible with the following deep sequencing library construction. Following PNK treatment, the ribosome-protected mRNA fragments (RPFs) were purified by VAHTS RNA Clean Beads followed by library construction with VAHTS Small RNA Library Prep Kit for Illumina (Vazyme, NR801). The libraries were fractioned on 6% PAGE minigel and the bands ranging around 150bp were recovered and purified for quality control. The qualified libraries were sequenced on Illumina Nova Seq 6000.