title: Sample 8
organism: Homo sapiens
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-11-29T00:23:55Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-11-29T00:23:55Z
attributes.tag: External Id
attributes.value: SAMEA112328372
attributes.tag: INSDC center name
attributes.value: INSTITUT CURIE
attributes.tag: INSDC first public
attributes.value: 2023-11-29T00:23:55Z
attributes.tag: INSDC last update
attributes.value: 2023-11-29T00:23:55Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12501:Sample 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell cycle stage
attributes.value: G1/S synchronized
attributes.tag: cell_line
attributes.value: HeLa
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: adenocarcinoma
attributes.tag: fraction
attributes.value: total RNA
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: mixed uterus and cervix
attributes.tag: sample name
attributes.value: E-MTAB-12501:Sample 8
attributes.tag: scientific_name
attributes.value: Homo sapiens