Ribosome profiling following AZD1775 10 hour treatment in RPE1 TP53- cells

RPE-1 TP53-/- cells were treated with either DMSO or 650 nM AZD1775 for 10 hours (biological n=3). Following the incubation, the growth media was aspirated and washed in ice-cold PBS/CHX (1 X PBS supplemented with 100 µg/mL cycloheximide). Following this, 3 mL of ice old PBS/CHX was added per dish, cells were scraped extensively and pelleted at 300G centrifugation. Supernatant was aspirated and the pellets were snap frozen. Flash frozen cell pellets were lysed in ice-cold polysome lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100) supplemented with cycloheximide (100 µg/mL). For ribosome profiling (Ribo-seq), remaining lysates were digested in the presence of 35U RNase1 for 1 hour at room temperature. Following RNA purification, PNK end repair and size selection of ribosome protected mRNA fragments on 15% urea PAGE gels, contaminating rRNA was depleted from samples using EIRNABio's custom biotinylated rRNA depletion oligos. Enriched fragments were converted into Illumina compatible cDNA libraries. Ribo-seq libraries were sequenced on Illumina's Nova-seq 6000 platform (single read) to depths of 100 million raw read pairs per sample.