Ribosome profiling

To analyze ribosome-contained mRNA fragments, steps such as RNase I treatment, ribosome recovery, footprint fragment purification, 3′ adaptor ligation, rRNA depletion, reverse transcription, circularization, and PCR amplification were performed essentially as described (21), with some modifications as described below. Brain or liver lysis was performed by transferring each frozen left half-brain or liver into polysome buffer (20) and homogenizing using TissueRuptor (QIAGEN). For 3′ adaptor ligation, air-adenylated linker A (BIOO, 5rApp/CTGTAGGCACCATCAAT/3ddC/) was used. cDNA libraries of ribosome-contained mRNA fragments were initially sequenced using MiSeq to perform A site codon analysis using Galaxy (53) and RiboGalaxy (54): First, fastq files were filtered by quality, the 3′ adaptor sequence (CTGTAGGCACCATCAAT) was removed, and rRNA reads were removed by Bowtie2. Ribosome-contained mRNA fragment sequences were acquired by aligning reads to mouse RefSeq mRNA sequences and were analyzed using triplet periodicity and metagene analysis programs in RiboGalaxy, to determine the appropriate footprint length and codon frames. For large-scale ribosome-contained mRNA fragment analysis, cDNA libraries were subjected to Illumina HiSeq2500 sequencing. Using Galaxy, fastq files were filtered by quality, the adaptor sequence (CTGTAGGCACCATCAAT) was removed, and mRNA reads were retrieved by aligning to mouse RefSeq mRNAs. mRNA reads were aligned, counted, and analyzed using HISAT2, HTSeq-count, and DESeq2. Gene ontology analysis was performed using Gene Set Enrichment Analysis (GSEA) website software (55).