Artseq

For ribosome footprint profiling experiments, 1 × 10⁹ platelets were treated with cycloheximide (100 mg/ml) for 1 min at RT to preserve ribosomes as natively attached to mRNAs.^{35 (link)} The platelets were lysed in Mammalian Lysis Buffer (ARTseq, Epicentre). Total RNA and ribosome-protected read RNAs (RPRs) were prepared per the manufacturer’s instructions (ARTseq-R ibosome Profiling Kit, Epicentre). Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD- 401– 4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC- 401– 4002). Ribosome profiling (eukaryote) library preparation was completed using the NEBNext Multiplex Small RNA Library Prep Set.