Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: At each time point, cells were placed on ice, washed with 10 mL ice-cold PBS with 100 μg/mL CHX, and lysed in plate in 400 uL of cold lysis buffer (20 mM Tris, pH.4; 150 mM NaCl; 5 mM MgCl2; 1 mM DTT; 1% Triton X-100; 50 μg/mL emetine; 25 U/mLTurbo DNase [Thermo Fisher Scientific]). Cells were then scraped off the plate, transferred to a 1.5 mL tube, and incubated on ice for 5 min. Cell debris was pelleted at 16,000 x g for 10 min at 4°C. The resulting supernatant was then snap frozen in liquid nitrogen before storing at -80°C. Ribosome profiling was performed as previously described (Ingolia et al. 2012) with minor modifications. Briefly, cell lysates were thawed, treated with RNase I, and then pelleted over a 1 M sucrose cushion containing 100 μg/mL emetine. RNA was then size-selected by excising between the 15 and 34 nt marker oligonucleotides, and ligated to the Universal miRNA Cloning Linker (NEB). rRNA depletion was then performed using the RiboZero rRNA Removal Kit (Human version, Illumina), followed by reverse transcription, circularization, and PCR amplification. For RNA-seq, total RNA was extracted from thawed cell lysates. Libraries were then made from 0.3 μg of total RNA using the TruSeq Stranded Total RNA kit (Illumina) according to the manufacturer's instructions, and amplified by PCR for 10 cycles. All libraries were sequenced on a HiSeq 2500 system at the Genetic Resource Core Facility of the Johns Hopkins University School of Medicine