2024-12-01
ENA first public
ERC000011
ENA-CHECKLIST
Université Paris Cité, INSERM U1151, CNRS UMR8253, Institut Necker Enfants Malades
INSDC center name
public
INSDC status
ERS20128798
SRA accession
E-MTAB-14119:NS-1
Submitter Id
ArrayExpress
broker name
HCT116
cell_line
colorectal cancer cell
cell_type
not collected
collection date
human
common name
Protocols: Samples were washed with PBS and lysed directly in the dish with TRIzol.  HCT116 human colorectal cancer cells were plated 2 days before infection in 6-well plates for an approximate confluence of cells of 70% on the day of infection. Cells were infected with Shigella flexneri (strain M90T, MOI 100) previously grown into late exponential face (OD=0,7). Plates of cells with bacteria were centrifuged 10min at 300g and incubated 30min at 37°C, 5% CO2 and in humid atmosphere for invasion of cells by the bacteria. Extracellular bacteria were washed away by 3 PBS washes of the infected cells. Cells were then incubated in basal DMEM medium supplemented with 50µg/ml gentamicin for 5h in 37°C, 5% CO2 humid atmosphere.  Total RNA was purified by TRIzol (Life Technologies, 15596026) using 1ml of reagent for each 6 million of cells or less, according to the manufacturer’s instructions. RNA samples were then subjected to DNase digestion (DNase TURBO, Life technologies), followed by acidic phenol/chloroform purification.  From the total RNA samples, ribosomal RNA molecules were depleted using the Ribo-Zero rRNA Removal Kit (Illumina) using a 1:1 ratio of probes from the Human/Mouse/Rat and Bacteria module.The rRNA depleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase (13 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).
description
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
non infected control
infect
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
colon
organism part
Homo sapiens
scientific_name
NS-1
title