GSM8010375: A549 cells, Mock, Ribo-Seq, rep 1; Homo sapiens; OTHER
Sample was treated with specific lysis buffer with cycloheximide (50mg/mL) to acquire the lysate. To digest the RNA other than ribosome-protected fragments (RPFs), cell lysate was treated with unspecific endoribonuclease RNase I. Isolation of monosomes was performed by size-exclusion chromatography with MicroSpin S-400 HR columns. Following PAGE purification, the both ends of RPF were phosphorylated and ligated with 5' and 3' adapters respectively. The RNA samples were then treated with rRNA depletion kit（Qiagen） to deplete the samples of as much rRNA contamination as possible before PAGE purification of the relatively short (20~38 nt) RPFs. Then the fragments were reversely transcribed to the cDNAs and amplified by PCR.