GSM8415496: WPMY-1, shcontrol-1,ribo-seq; Homo sapiens; OTHER
Total RNA was extracted using Trizol reagent In order to digest the RNA other than ribosome-protected fragments (RPFs or RFPs), cell or tissue lysate was treated with unspecific endoribonuclease RNase I. Monosomes were purified from the lysateby fractionation on a linear sucrose gradient. 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 (25~35nt) RPFs. Then the fragments were reversely transcribed to the cDNAs and amplified by PCR. After library construction, the concentration of library was measured by Qubit® 2.0 Fluorometer and adjusted to 1ng/μL. Agilent 2100 Bioanalyzer was deployed to examine the insert size of the acquired library. At last, the accurate concentration of cDNA library was again examined using qPCR. Once the insert size and concentration of the library was correct, the samples can then be subjected for sequencing.