footprint.replicate2.timepoint.2
Library strategy: ribosome profiling. Total RNA was collected from the cell lysate using Trizol-chloroform extraction and elution with water. Ribosome-bound RNA was generated by treating the lysate with RNAse I and quenching the reaction with Superase-In RNase Inhibitor, then isolating ribosomes using Sephacryl MicroSpin columns. Genomic DNA was removed by DNase I digestion. We prepared barcoded libraries for sequencing using the NEBNext Small RNA Library Prep Set for Illumina for the ribosome-bound fragments. Libraries were pooled, denatured and diluted according to the NextSeq 500 protocol. Single-end sequencing of libraries was performed on the Illumina Nextseq 500 platform using high-output flowcells with 75 bp read lengths.