GSM7426257: Ribo ShControl rep 1; Homo sapiens; OTHER
To prepare ribosome-protected mRNA fragments, 10 μl of RNase I and 6 μl of DNase I were added to the supernatant and incubated at room temperature for 45 min, followed using 10 μl RNase inhibitor (Thermo Scientific, #AM2694) to stop the nuclease digestion. Size exclusion columns (GE Healthcare, #27-5140-01) were equilibrated with 3 ml of polysome buffer, and digested RNAs were added to the column and centrifuged at 600 g for 2 min. Next, 10 μl 10% SDS was added to the elution, and the ribosome-protected mRNA fragments with a size greater than 17 nt were isolated according to the RNA Clean and Concentrator-25 kit (Zymo Research, #R1017) and further purified using magnet beads (Vazyme, #N412-01). Ribo-seq libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (New England Biolabs, #E7300L)