HEK293T Mtr4-KD and Control Ribo-Seq Reads for Ribosome Profiling

Ribo-seq reads from Mtr4-knockdown and siRNA control cells used for ribosome profiling under Mtr4-depleted conditions. Cell line: HEK293T Transfection: siRNA, 72 hours Cells were lysed with cycloheximide lysis buffer, ribosomes were isolated by Ambion RNaseI digestion of unprotected RNA followed by sucrose gradient fractionation. Footprints were obtained by TRIZOL-chloroform extraction from ribosome fractions followed by urea-PAGE gel extraction. Libraries were made using the NEBNext Small RNA Library Prep Kit and rRNA fragments were depleted with custom biotinylated oligos. Sequencing was performed using an Illumina NextSeq 500 for 400 million single-end reads. Reads were converted into FASTQ format by Illumina BaseSpace. Adapters were removed by CutAdapt, remaining rRNA reads were removed by Bowtie2 alignment vs published rRNA sequences.