GSM2476122: HEK293 RPF Rep2; Homo sapiens; OTHER
HEK293 cells were treated with cycloheximide (CHX, Sigma-Aldrich, final concentration 0.1mg/ml) for 1 minutes. In-dish cell lysis were performed using Mammalian Lysis Buffer (including CHX at a concentration of 0.1mg/ml). Then 600 μl of lysate were taken and 15 ul of RNase I (100 U/μl, Thermo Fisher Scientific) were added and the mixtures were incubated for 45min at room temperature followed by adding 15 μl SUPERaseIn RNase inhibitor (Thermo Fisher Scientific) to stop the reaction. Ribosome recovery were done by illustra MicroSpin S-400 HR Columns (GE Healthcare) and the Ribosomal Protected Fragments (PFFs) were purified by RNA Clean & Concentrator (Zymo Research). Ribosomal RNA was subtracted using Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat, Illumina). RPFs without ribosomal RNA were ran on a 15% Urea denaturing-PAGE gel, and the gel slides from 28nt~30nt were excised. The RPF RNA were eluted and precipitated followed by library construction according to the Manufacturer’s protocol.
GEO Accession
GSM2476122