GSM2100600: M phase ribosome profiling synchronized by shake off; Homo sapiens; OTHER
Cells were lysed, treated with RNaseI, purified by spin-column and RNAs were extracted by TRIzol (Invtirgen) Ribosome profiling libraries were prepared using ARTseq Ribosome Profiling Kit (Epicentre) with some modifications. HeLa cells were grown in two 150 mm dishes, harvested and lysed with lysis buffer. For nuclease digestion, 250 μl of cell lysates were treated with 3 μl of ARTseq nuclease at 4° C for 45 min at RT. Monosomes were purified using MicroSpin S-400 column by centrifuging for 2 min at 600 g. RNAs in the flow-through were purified and depleted of rRNAs using Ribo-Zero kit (Epicentre). rRNA depleted RNA fragments were then radiolabeled, and ~30 nt region was purified using 15% Urea-PAGE. Purified RNAs were treated with Antarctic phosphatase (NEB), 5′ phosphorylated and gel purified to remove free ATP. Resulting RNAs were subjected to 3′ adaptor ligation, and gel purified ligated products were ligated with the 5′ adaptor. Adaptor ligated RNAs were reverse transcribed and amplified to yield final ribosome profiling library. The cDNA libraries were sequenced by Illumina HiSeq (1 × 51 bp single run).
GEO
GSM2100600