GSM7816486: SupT1 infected, RiboSeq, harr, 24hpi, rep1; Homo sapiens; Human immunodeficiency virus 1; OTHER
Ribosome profiling samples were prepared as previously described (McGlincy and Ingolia., 2017) with modifications for suspension cell lines. Briefly, at each time point of infection, cells were treated with 100 μg/ml cycloheximide in DMSO at 37 °C for 5 min. For harringtonine treated samples, cells were treated with 2 μg/ml harringtonine in DMSO at 37 °C for 5 min, followed by cycloheximide treatment. Cells were immediately pelleted at 1000 RPM for 3 min at 4 °C, washed once with cold cycloheximide containing PBS and the cell pellet was snap-frozen in liquid nitrogen and stored at -80 °C, till further use. Cell pellet was thawed in the presence of ice-cold lysis buffer (20 mM Tris-Cl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, 1% Triton-X, 25U/ml Turbo DNase ) and triturated 10 times through a 26G needle. The lysate was centrifuged at 17000g for 10 min at 4 °C and the supernatant was recovered. Cell lysates were subjected to RiboSeq and RNASeq. RiboSeq libraries: RNase I footprinting of ribosomes, ribosome purification by ultracentrifugation through a sucrose cushion, RNA extraction, polyacrylamide gel size selection for footprint fragments, preadenylylated linker ligation, reverse transcription, first-strand cDNA circularization. RNASeq libraries from these samples were constructed using CORALL Total RNA-Seq Library Prep Kit (Lexogen), according to manufacturer's instructions.