ERC000011
ENA-CHECKLIST
2023-11-29T00:23:55Z
ENA-FIRST-PUBLIC
2023-11-29T00:23:55Z
ENA-LAST-UPDATE
SAMEA112328365
External Id
INSTITUT CURIE
INSDC center name
2023-11-29T00:23:55Z
INSDC first public
2023-11-29T00:23:55Z
INSDC last update
public
INSDC status
ERS14439280
SRA accession
E-MTAB-12501:Sample 20
Submitter Id
ArrayExpress
broker name
G1/S asynchronous
cell cycle stage
HeLa
cell_line
human
common name
Protocols: For synchronized cells, a double thymidine block has been applied. For the 4sU-labeled RNA-seq, cells were cultured in 15 cm dish and 10 ml of new medium containing 1 mM 4sU was added for 8 minutes (Fuchs et al., 2015). HeLa cells were grown in DMEM 1X 4.5 g/L D-Glucose, L-Glutamine, Pyruvate (Gibco) with 10% Fetal Cow Serum FCS (Eurobio) and 1% Penicillin/Streptomycin (Gibco). Cells were transfected with siRNA at 100 nM concentration using Lipofectamine RNAiMAX (Invitrogen, 13778-150) as previously shown in (Clément et al., 2018) and for plasmids using Lipofectamine 2000 (Invitrogen, 11668-019). Cells were lysed, and total RNA was extracted directly from the cell culture dishes using the RNeasy Mini Kit according to the manufacturer’s instructions. The total RNA was DNase treatment using the TURBO DNA-free kit (ThermoFisher Scientific, AM1907) using manufacturer’s instructions. The quantity of isolated RNA was measured using the Nanodrop and quality was checked by Tapestation. For 4sU-labeled RNA purification, 100-200 μg RNA was used for RNA biotinylation and purification was performed using Streptavidin-coupled Dynabeads (Fuchs et al., 2015). 10 ng of total and 4sU-labeled RNA was used for library preparation using SMARTer Stranded Total RNA-seq kit.
description
adult
developmental stage
adenocarcinoma
disease
nascent RNA
fraction
wild type genotype
genotype
Homo sapiens
organism
Homo sapiens
organism
mixed uterus and cervix
organism part
Homo sapiens
scientific_name
Sample 20
title