title: GSM9376078: HAP1 cells, aza-C + GCN2i  treated, disome, biol rep1; Homo sapiens; OTHER
library_construction_protocol: After treatment cells were washed with ice-cold PBS supplemented with 10 mM MgCl2 and 800 µM cycloheximide. Cells were scraped in 100 µL 5x lysis buffer (0,25 M HEPES (KOH) pH 7.5, 50 mM MgCl2, 1 M KCl, 5% (vol/vol) NP40, 1000 μM cycloheximide) on ice. Lysates were vortexed and clarified by centrifugation for 10 min at 20,000 xg and 4°C. For 120 ng RNA in the lysate, 100 U RNase I were added and incubated on ice for 5 min. Subsequently the lysates were loaded onto 10-50% sucrose density gradient (10 mM Tris pH 7.4, 10 mM MgCl2, 160 mM KCl) and centrifuged in an SW40Ti rotor (Beckman Coulter) at 202,408 x g for 165 minutes. Fractionation was performed using a BioComp Gradient Station and absorption profiles at 260 nm were recorded using a Triax Flow Cell FC-2. Monosome and disome fractions were collected. RNA extraction and size selection (25-35 nt for monosome footprints and 54-68 nt for disome footprints) was performed as decribed previsously (Wagner, Bohlen, Herrmannova et al., 2022). rRNA depletion and library preparation were performed as decribed previsously (Wagner, Bohlen, Herrmannova et al., 2022).