2025-05-31
ENA first public
ERC000011
ENA-CHECKLIST
Laboratory of Molecular Medicine and Genomics - University of Salerno
INSDC center name
public
INSDC status
ERS20849776
SRA accession
E-MTAB-14328:202_FSK_poly
Submitter Id
ArrayExpress
broker name
U87MG
cell_line
not collected
collection date
human
common name
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
description
polysomes
fraction
FSK
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
Homo sapiens
scientific_name
202_FSK_poly
title