GSM9154507: H358 cells, shControl, Ribo-Seq,  rep2; Homo sapiens; OTHER
For Ribo-Seq,  Cells were washed twice with ice-cooled PBS containing 100 μg/mL CHX, and lysed. Lysates were incubated on ice for 10 min, followed by centrifugation at 16000 x g for 10 min at 4°C. A portion of supernatants was saved for RNA-Seq. Ribo-Seq samples were digested with MNase or RNAse I (Epicenter) and digested polysomes were loaded on a 10-50% sucrose density gradient and centrifuged at 36,000 rpm for 3 hours at 4°C. Fractions corresponding to monosome peak fractions were collected, pooled, and RNAs were extracted using Trizol. For Ribo-Seq library prep, we followed McGlincy and Ingolia 2017 ribosome profiling technique with some modification. RNA was electrochemically separated on a 15% TBU-Urea gel, and fragments of 25-40 nt were selected. After gel extraction, rRNA depletion, and dephosphorylation, ligation was performed with pre-adenylated linkers containing Unique Molecular Identifiers (UMIs) and barcodes. The ligated samples underwent gel purification, reverse transcription, and circularization using CircLigase I. Another rRNA depletion step was done before qPCR optimization. The final PCR product, confirmed for size and quantity, was sequenced on an Illumina NovaSeq 6000 System.