GSM8721423: siDDX24_1_RiboSeq_rep3; Homo sapiens; OTHER
HUVEC cells were transfected with DDX24-specific siRNA or negative control siRNA for 48 hours. For cell lysis preparation, the culture medium was removed, and cells were washed with ice-cold PBS containing cycloheximide (CHX, 100 µg/mL; MP Biomedicals 0219452701). The ribosome profiling assay was conducted following previously described protocols with modifications. Cells were lysed in ice-cold mammalian polysome buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, supplemented with 1 mM DTT, 1% Triton X-100, 0.1% NP-40, 500 U/mL RNase Inhibitor, 1× EDTA-free Complete protease inhibitors, 1 U/µL DNase I, and 100 µg/mL cycloheximide. The lysate was homogenized by passing through a 26-gauge needle ten times and clarified by centrifugation (20,000×g, 5 minutes, 4°C). Ten percent of the cell lysate was reserved as an input control for RNA sequencing after mRNA enrichment. The supernatant (120 µL) was treated with RNase I (3 µL; Life Technologies, AM2294) for 45 minutes at room temperature. The reaction was terminated by adding SUPERase•In RNase Inhibitor (Life Technologies, AM2694). Monosomes were purified using MicroSpin S-400 HR columns (GE Healthcare, 27-5140-01), and RNA was isolated from the flow-through (approximately 125 µL) using RNAzol (1 mL). Ribosome footprints (RFPs) were enriched by depleting ribosomal RNA fragments using biotin-labeled probes specific for rRNA sequences. The remaining RNA was separated on a 17% 7.5M Urea-PAGE gel, and fragments between 26-30 nucleotides were excised using a 14-30 ssRNA Ladder Marker (Takara, #3416) as reference. For the input control, RNA was isolated using RNAzol, treated with DNase I, and enriched for mRNA using Oligo-dT beads (NEB, E7490). The polyA mRNA was then fragmented, and 30-50 nucleotide fragments were recovered from the Urea-PAGE gel. Before library construction, both ribosome-protected fragments (RPFs) and input fragments were subjected to T4 polynucleotide kinase treatment for end repair. The libraries were prepared following the protocol previously described in Nature Methods and were sequenced on the HiSeq X Ten platform at Annoroad.