2025-02-27
ENA first public
ERC000011
ENA-CHECKLIST
University of Salerno
INSDC center name
public
INSDC status
ERS20212303
SRA accession
E-MTAB-14147:IgG_1
Submitter Id
ArrayExpress
broker name
MCF-7
cell_line
mammary epithelial cell
cell_type
not collected
collection date
human
common name
Protocols: Cells were washed twice with cold PBS supplemented with 0.1% of EDTA (500 mM), harvested by scraping and collected in a tube. After a centrifugation at 3000 rpm for 5 min at 4°C, the nuclear fraction was extracted by resuspending the pellet in nuclear isolation Buffer (NIB) (1.28 M Sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2 and 4% Triton X-100) supplemented with 100 U/ml RNAse inhibitor (RiboLock RNase Inhibitor, Invitrogen) and proteinase inhibitors (1mM PMSF and 1x PIC). Samples were incubated on ice for 20 min and then centrifuged at 2500 x g at 4°C for 15 min. Once removed the supernatant, nuclear pellets were resuspended in 400 µl of RIP Buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT e 0.5% NP40) supplemented with 100 U/ml RNAse and proteinase (1mM PMSF and 1x PIC) inhibitors. Cell nuclei were sonicated for 10 cycles (15 ON and 15 OFF) using Bioruptor (Diagenode, Denville, New Jersey, USA) and subsequently centrifuged at 13000 rpm for 10 min at 4°C. 1 ml of TRIzol (Life Technologies, Thermo Fisher) was added directly to the beads and RNA extraction was performed according to the manufacturer's guidelines TruSeq Stranded Total RNA Library Prep Gold (Cat. 20020599, Illumina, San Diego, California, USA)
description
adult
developmental stage
breast neoplasm
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
breast
organism part
Homo sapiens
scientific_name
IgG_1
title