title: GSM9503283: ID8, WT-2; Mus musculus; OTHER
library_construction_protocol: Cells were harvested after treatment with 100 µg/mL cycloheximide for 2 minutes to arrest ribosomes. Cell lysates were prepared in lysis buffer with cycloheximide and RNase inhibitors. Ribosome-protected fragments (RPFs, ~28-30 nt) were generated by digesting lysates with RNase I, followed by ribosome isolation via sucrose cushion centrifugation. RPFs were size-selected by gel purification, dephosphorylated, and ligated to a pre-adenylated linker for library construction. Raw sequencing reads were trimmed of adapter sequences using Cutadapt. Reads with lengths between 25-34 nt were retained. rRNA and tRNA reads were removed by alignment to rRNA/tRNA sequences using Bowtie. Remaining reads were aligned to the reference genome (GRCm38/mm10 for mouse) and transcriptome using HISAT2 and Bowtie, respectively.