GSM9541164: Disome_K-A; Homo sapiens; OTHER
For mRNA reporters, 8 μg of mRNA was transfected into HEK293-Kb cells (~80% confluence) in 10 cm dishes for 8 h. For plasmid reporters, 1 μg plasmid was transfected into 35 mm dishes for 24 h. Cells were washed with cold PBS and lysed in polysome lysis buffer (10 mM HEPES, pH 7.4; 100 mM KCl; 5 mM MgCl₂; 100 μg/mL cycloheximide; 1% Triton X-100). Lysates were cleared by centrifugation at 20,000 × g for 10 min at 4 °C and digested with RNase I (Ambion, 750 U per 100 A₂₆₀ units) at 4 °C for 1 h. Total RNA was extracted using TRIzol LS (Invitrogen) for cDNA library construction. Ribosome-protected fragments (25–35 nt) or disome footprints (40-60 nt) were resolved on a 15% polyacrylamide TBE–urea gel (Invitrogen) and visualized with SYBR Gold. Excised RNA fragments were eluted overnight in RNA elution buffer (300 mM sodium acetate, pH 5.2; 1 mM EDTA; 0.1 U/μL SUPERase·In), cleared through Spin-X columns, and ethanol-precipitated. cDNA libraries were constructed using T4 PNK, Poly(A) polymerase, and custom Ezra enzyme, followed by ligation of 5' adapters, streptavidin bead-based purification, and reverse transcription following the previous study [28]. Final cDNA was amplified with barcoded primers, size-selected (180 bp), quantified, pooled, and sequenced on an Illumina MiniSeq or NextSeq 500.