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
ERS20120808
SRA accession
E-MTAB-14115:NTC4_DOX
Submitter Id
ArrayExpress
broker name
HeLa
cell_line
epithelial
cell_type
not collected
collection date
human
common name
Protocols: Cells were stimulated (or not) with 100ng/ml doxycycline for 48h to induce microRNA expression. Three independent stimulations were performed.  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 A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) or by using sonication with Diagenode bioruptor Pico for breaking RNA strands. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase （RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB， USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
description
adult
developmental stage
adenocarcinoma
disease
NTC (piSMART mCMV/TurboGFP non-targeting control)
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
uterus
organism part
Homo sapiens
scientific_name
NTC4_DOX
title