organism: Homo sapiens
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-03-31T17:01:54Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-05-30T11:46:20Z
attributes.tag: External Id
attributes.value: SAMEA4699308
attributes.tag: INSDC center name
attributes.value: Medical University of Gdansk Department of Biology and Pharmaceutical Botany
attributes.tag: INSDC first public
attributes.value: 2019-03-31T17:01:54Z
attributes.tag: INSDC last update
attributes.value: 2018-05-30T11:46:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6832:BR.1N zebularine
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HaCaT
attributes.tag: cell type
attributes.value: keratinocyte
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: normal
attributes.tag: organism part
attributes.value: skin
attributes.tag: sample name
attributes.value: E-MTAB-6832:BR.1N zebularine
attributes.tag: scientific_name
attributes.value: Homo sapiens