characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-10-02T00:46:12Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-10-02T00:46:12Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110758621
characteristics: External Id
characteristics: IOCB Prague
characteristics: INSDC center name
characteristics: 2022-10-02T00:46:12Z
characteristics: INSDC first public
characteristics: 2022-10-02T00:46:12Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12856231
characteristics: SRA accession
characteristics: E-MTAB-12053:Imm. Primary + LACTB DAY 10_1
characteristics: Submitter Id
characteristics: over 18
characteristics: year
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: ovarian epithelial cell
characteristics: cell_type
characteristics: human
characteristics: common name
characteristics: Protocols: The primary and PEO4 cells were purchased. Primary (Cell biologics H-6036) andPEO4 (Sigma Aldrich 10032309-1VL) All cells were cultured in 5% CO2 humidified incubator at 37 °C. To activate the tetracycline-inducible gene expression, the cells were treated with 1 μg/ml DOX hyclate (Sigma-Aldrich) in medium. Fresh DOX was added every 3 days. PEO4 and PRIMARY were cultivated with RPMI 10%FBS and 1% P/S. The Primary cells were immortalized in our laboratory through the addition of hTERT. Immortalization protocol: 1. Plate the target cells in one 10cm plate of 1x106 cells. 2. The next day, infect the target cells with hTERT virus. 24h incubation at 37˚C 3. After 48 hours incubation, start the selection with HYGRO. Total RNA was isolated with GenElute™ Total RNA Purification Kit (RNB100-100RXN) and subjected to RNA-sequencing core facility at Biocev, Institute of Molecular Genetics, Czech Republic. Total RNA was isolated with GenElute™ Total RNA Purification Kit (RNB100-100RXN) and subjected to RNA-sequencing core facility at Biocev, Institute of Molecular Genetics, Czech Republic. For the results analysis the read quality was assessed using FastQC 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapters and low-quality sequences were removed using Trimmomatic 0.39 (DOI 10.1093/bioinformatics/btu170). Clean reads were then mapped into the hg38 human genome using HISAT 2.1.0 (DOI 10.1038/s41587-019-0201-4) and Samtools 1.10 (DOI: 10.1093/gigascience/giab008). Read coverage profiles were then computed using the bamCoverage tool from the deepTools 3.5.1 package (DOI 10.1093/nar/gkw257). Mapped reads and coverage data were inspected visually in the IGV 2.9 browser (DOI 10.1038/nbt.1754). Differentially expressed genes were detected using the DESeq2 package (DOI 10.1186/s13059-014-0550-8) in R/Bioconductor (DOI 10.1186/gb-2004-5-10-r80; DOI 10.1038/nmeth.3252). All scripts and code are available from GitHub at https://github.com/mprevorovsky/keckesova-LACTB. Heatmap was generated from the output of DESeq2 analysis performed on the RNA-seq data, with genes considered statistically significant (P < 0.05). Z-scores were computed from the normalized gene counts.
characteristics: description
characteristics: adult
characteristics: developmental stage
characteristics: normal
characteristics: disease
characteristics: LACTB +/+; TetO hTERT
characteristics: genotype
characteristics: Homo sapiens
characteristics: organism
characteristics: Homo sapiens
characteristics: organism
characteristics: ovary
characteristics: organism part
characteristics: Homo sapiens
characteristics: scientific_name
characteristics: female
characteristics: sex
characteristics: Imm. Primary + LACTB DAY 10_1
characteristics: title