GSM8912446: Ribo-Tre-rep2; Homo sapiens; OTHER
For ribo-seq，Sample was treated with specific lysis buffer with cycloheximide to acquire the lysate, cell lysate was treated with unspecific endoribonuclease RNase I. Monosomes were purified from the lysateby fractionation on a linear sucrose gradient. For RNA-seq, total RNA was extracted from cells using RNA isolater Total RNA Extraction Reagent. For Ribo-seq, 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, Germany) to deplete as much rRNA contamination as possible before PAGE purification of the relatively short (25~36nt) RPFs. Then the fragments were reversely transcribed to the cDNAs and amplified by PCR. Concentration of the cDNA was measured by Qubit® 2.0 Fluorometer. After library construction（Multiplex Small RNA Library Prep Set for ILLumina (Set1)，NEB）, the concentration of library was measured by Qubit® 2.0 Fluorometer. Agilent 2100 Bioanalyzer was deployed to examine the insert size of the acquired library. The cDNA library products were generated using VAHTS Small RNA Library Prep Kit.; RNA libraries for RNA-seq were prepared using ployA enrichment method following manufacturer's protocol