GSM7038005: NEP-2 Ribo-seq; Homo sapiens; OTHER
The hESC, NEP and ME cells were treated with CHX (100 ug/ml, Sigma, St Louis, MO, USA) for 5 min to freeze the translating ribosomes. The ribosomal profiling technique was carried out as reported previously, with a few modifications as described below. 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 20000 g for 10 min at 4 °C, and the supernatant was collected. To prepare RFs, 10 µl of RNase I (NEB, Ipswich, MA, USA) and 6 µl of DNase I (NEB, Ipswich, MA, USA) were added to 400 µl 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 10 µL of SUPERase·In RNase inhibitor (Ambion, Austin, TX, USA). Size exclusion columns (illustraMicroSpin 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 600 g for 4 min at room temperature. 100 μL of digested RFs were added to the column and centrifuged at 600 g for 2 min. Next, 10 μL 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). The rRNA was removed using the method reported previously. Briefly, 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 140-160 bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeqTM X10.