GSM7747668: AC16 cells, AGO2-KO, 1; Homo sapiens; OTHER
Cells pellet were immediately frozen in liquid nitrogen and ground to power in liquid nitrogen with a mortar and pestle, then dissolved in 400μL of lysis buffer. The resuspended extracts in lysis buffer were transferred to new microtubes, pipetted several times and incubated on ice for 10 min. Then cells were triturated ten times through a 26-G needle. The lysate was centrifuged at 20000g for 10min at 4℃, and the supernatant was collected. To prepare RFs , 10μL of RNase I (NEB, Ipswich, MA, USA) and 6μL of DNase I (NEB, Ipswich, MA, USA) were added to 400μL of lysate，which was then incubated for 45 min at room temperature with gentle mixing on a Nutator mixer. Nuclease digestion was stopped by adding 10μL of SUPERase·In RNase inhibitor (Ambion, Austin, TX, USA). Size exclusion columns (illustra MicroSpin S-400 HR Columns; GE Healthcare; catalog no. 27- 5140-01) were equilibrated with 3 mL of polysome buffer by gravity flow and centrifuged at 600g for 4 min at room temperature. 100μL of digested RFs were added to the column and centrifuged at 600g for 2 min. Next, 10μL 10% (wt/vol) SDS was added to the elution, and RFs with a size greater than 17nt was isolated according to the RNA Clean and Concentrator-25 kit (Zymo Research;R1017). short (50–80 bases) antisense DNA probes complementary to rRNA sequences was added to solution containing RFs, then RNase H (NEB, Ipswich, MA, USA) and DNase I (NEB, Ipswich, MA, USA) was added to digest rRNA and residual DNA probes. Finally, RFs were further purified using magnet beads (Vazyme, Nanjing, Jiangsu, China). After obtaining ribosome footprints above, Ribo-seq libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (catalog no.E7300S, E7300L). Briefly, adapters were added to both ends of RFs, followed by reverse transcription and PCR amplification. The 140-160bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeqTM X10 by Gene Denovo Biotechnology Co. (Guangzhou, China).