BR.1N zebularine
Homo sapiens
Protocols: Cultured keratinocytes (HaCaT) and fibroblasts (46BR.1N) for RNAseq analysis were trypsynised, washed, and snap frozen for transcriptomic analysis. The pellets were stored in - 70ºC prior to RNA isolation. Immortalized human keratinocytes (HaCaT, (DKFZ Heidelberg, Germany) and human, dermal fibroblasts (46BR.1N, Sigma-Aldrich) were grown in DMEM-HG (Sigma-Aldrich, D6429) supplemented with 10% of fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. The cells were routinely cultured under a humidified atmosphere with 5% CO2 at 37°C. For zebularine stimulation, the medium was exchanged for DMEM supplemented with 2% FBS, 16 μM thymidine, and zebularine (1 μg/mL). After 48 h, medium was exchanged and cells were stimulated with zebularine for a second time. After another 48 h of incubation, cells were detached, counted, washed in PBS, centrifuged and stored as pellets at -80°C. Three independent cell culture experiments were performed for each cell line. Total RNA was isolated from cultured keratinocytes (HaCaT) and fibroblasts (46BR.1N) using miRNeasy Mini Kit (Qiagen) according to the manufacturer's instruction except for that 1-bromo-3-chloropropan was used for extraction. RNAs extracted from cell culture replicates were pooled in equal concentrations. The measured RIN values were >8.0. Ribosome reduction kit (Arraystar Inc, Rockville, USA) and SureSelect Strand Specific mRNA library kit (Agilent, Santa Clara, USA) were used according to standard procedure. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Library Quantification Kit for Illumina (Kapa Biosystems, Woburn, USA).
ENA-FIRST-PUBLIC
2019-03-31T17:01:54Z
ENA-LAST-UPDATE
2018-05-30T11:46:20Z
External Id
SAMEA4699308
INSDC center name
Medical University of Gdansk Department of Biology and Pharmaceutical Botany
INSDC first public
2019-03-31T17:01:54Z
INSDC last update
2018-05-30T11:46:20Z
INSDC status
public
Submitter Id
E-MTAB-6832:BR.1N zebularine
broker name
ArrayExpress
cell line
HaCaT
cell type
keratinocyte
common name
human
disease
normal
organism part
skin
sample name
E-MTAB-6832:BR.1N zebularine
scientific_name
Homo sapiens