2025-05-31
ENA first public
ERC000011
ENA-CHECKLIST
Laboratory of Molecular Medicine and Genomics - University of Salerno
INSDC center name
public
INSDC status
ERS20849817
SRA accession
E-MTAB-14331:IP_KO_FSK_esp1
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).  standard protocols for cell lines cells where treated with compound for certain number of hours. Cells were lysed in 1 mL of RNA immunoprecipitation (RIP) buffer (20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM DTT with freshly added 1:1000 protease inhibitor (Sigma) and 100 U of RNase inhibitor (20 U/µL, Thermo Fisher Scientific)) for 30 min on ice. Cell lysate was cleared by centrifugation at 15,000 g for 10 min at 4 °C. Subsequently, the supernatant was collected, and protein concentration was measured. 3 mg of extract were used for each IP in a final volume of 1 mL. 5 µg of DDX6 antibody and 5 µg of anti-IgG antibody (as a control) were added to the supernatant and incubated for two hours at 4 °C with gentle rotation. 50 μL of Dynabeads™ Protein A (10001D, Thermo Fisher Scientific) were washed two times with 0.5 mL of RIP buffer and then added to each sample, incubated for another two hours at 4 °C. The beads were collected and washed three times with 0.5 mL of RIP buffer; the last wash was performed with 0.5 mL of 1X PBS.  Then, 0.1% SDS (Sigma) and 50 μg proteinase K (20 mg/mL, AM2548, Thermo Fisher Scientific) were added to each sample and incubated for 30 min at 50 °C with 1100 rpm. DNA- and protein-free RNAs were collected from the beads using a magnetic stand. RNAs were further purified through RNA Clean and Concentrator-5 (R1013, Zymo Research) and eluted in 8 μL. Purified RNAs were used to make the cDNA libraries. Briefly, 150 ng of RNA of each input sample were used for library preparation as previously described, while an equal volume (5 ml) of each IP-purified RNAs were processed using the SureSelect XT HS2 mRNA Library Preparation System kit (Agilent Technologies) according to the manufacturer’s instructions. The resulting cDNA libraries were quantified by using the Qubit dsDNA BR Assay (Thermo Fisher) and analyzed by using the TapeStation chip (Agilent Technologies).
description
KO_FSK
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
Homo sapiens
scientific_name
IP_KO_FSK_esp1
title