Ribo-seq of Bos taurus_H3
Cells were treated with a specific lysis buffer containing cycloheximide (50 mg/mL) to obtain lysates. The cell lysates were treated with RNase I. Monosomes were isolated by size-exclusion chromatography using MicroSpin S-400 HR columns (GE Healthcare, Fairfield, CT, USA). Following polyacrylamide gel electrophoresis (PAGE) purification, both ends of RPFs were phosphorylated and ligated with 5' and 3' adapters, respectively. rRNA contamination was reduced using a GeneRead rRNA Depletion Kit (Qiagen, Hilden, Germany) before PAGE purification of the relatively short (approximately 2038 nt) RPFs. The fragments were then reverse-transcribed into cDNA and amplified using PCR.19, 20 A sequencing library was constructed using the Multiplex Small RNA Library Prep Set for Illumina (Set1; Illumina, San Diego, CA, USA). The insert size and library concentration were examined by the Agilent 2100 Bioanalyzer and The Qubit 2.0 Fluorometer, followed by sequencing. Ribo-seq was performed using SE50 (single-end 50 nt) sequencing of 6G raw reads on Illumina NovaSeq 6000 sequencer. Raw reads in fastq format were processed using in-house Perl scripts, and reads with 5' adapter, without 3' adapter or insert sequence, with > 10% N, with > 50% nucleotides with Qphred  20, with poly-A/T/G/C, with rRNA, and with tRNA were removed. Sequencing reads originating from rRNAs and tRNA were identified and discarded using Bowtie (version 1.1.2) and Bowtie (version 1.1.2), respectively. All downstream analyses were based on clean, high-quality data.