GSM9518380: Ribo-seq for RPSA; Homo sapiens; OTHER
Cells were lysed in buffer containing CHX, and total RNA was digested with low-concentration RNase I to degrade unprotected mRNA. Ribosome-protected fragments (RPFs, 26–35 nt) were purified by sucrose cushion ultracentrifugation, followed by phenol-chloroform extraction and ethanol precipitation. RPFs were dephosphorylated, 3′-adaptors were ligated, and 5′-phosphorylated adaptors were ligated to generate double-stranded cDNA. cDNA was amplified by PCR (12–15 cycles), size-selected (25–35 nt), and purified without gel extraction using SPRI beads. Library quality was assessed via Bioanalyzer (Agilent).