title: 201_WT_input
organism: Homo sapiens
description: Protocols: Media was aspirated from U87MG cells (two 150 mm plates per condition). Plates were immediately placed on ice and washed with ice-cold 1x phosphate-buffered saline (PBS) containing cycloheximide (100 μg/mL, Sigma-Aldrich). Cells were lysed in 800 μL of polysome extraction buffer (10 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 1% Triton X-100, 1 mM DTT, 10 U/mL RNaseOUT [Invitrogen], 100 μg/mL cycloheximide) and incubated on ice for 10 min. Lysates were cleared by centrifugation at 14,000 rpm at 4°C for 10 min. The supernatant was collected, and absorbance was measured at 260 nm using a NanoDrop. Between eight and ten optical density units were loaded onto a 10%–60% sucrose gradient formed by layering 6 mL of 10% sucrose over 6 mL of 60% sucrose prepared in polysome extraction buffer without Triton X-100 and containing 0.5 mM DTT, in a 12-mL tube (Polyallomer; Beckman Coulter). Gradients were prepared using a gradient maker (Gradient Master; Biocomp).  standard protocols for cell lines cells where treated with compound for certain number of hours. RNA extraction was performed by adding TRI reagent (Merck T9424) at a 1:1 v/v ratio to the fractions, followed by the manufacturer's protocol. Total RNA concentrations were determined by using NanoDrop spectrophotometer (Thermo Fisher) and their quality were assessed with the TapeStation 4150 (Agilent Technologies). Briefly, 150 ng of RNA of each sample were subjected to mRNAs capture by using polyT-coated beads followed by the indexed library preparation with the SureSelect XT HS2 mRNA Library Preparation System (Agilent Technologies), according to the manufacturer's instructions.  Polysomes were separated by centrifugation at 37,000 rpm for 2 hours using a Beckman SW41 rotor. Twelve fractions of 920 μL were collected, and polysomes were monitored by measuring absorbance at 254 nm
attributes.tag: ENA first public
attributes.value: 2025-05-31
attributes.tag: INSDC center name
attributes.value: Laboratory of Molecular Medicine and Genomics - University of Salerno
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-14328:201_WT_input
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: U87MG
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: fraction
attributes.value: Input
attributes.tag: genotype
attributes.value: WT
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: sample name
attributes.value: E-MTAB-14328:201_WT_input
attributes.tag: scientific_name
attributes.value: Homo sapiens