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Status |
Public on Jun 01, 2020 |
Title |
Control rep1 |
Sample type |
SRA |
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Source name |
U2OS
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Organism |
Homo sapiens |
Characteristics |
cell type: osteosarcoma epithelial cells cell line: U2OS genotype: Mock-transfected cells as control
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Treatment protocol |
In a six-well plate, approx. 80% confluent U2OS cells were transfected with various FOXP2 construct plasmids (500 ng) in equal amount using Lipofectamin 3000 (Thermo Fisher Scientific) for 48h. In case of CHIR-treatment, 5µM CHIR 99021 was added in the medium 72h before transfection with FOXP2 construct plasmids.
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Growth protocol |
Human Osteosarcoma Epithelial cells (U2OS) were maintained at 37°C under 5% CO2 in α-MEM medium (Invitrogen) supplemented with 5% (v/v) FCS (Gibco), 100 IU/ml penicillin and 100 µg/ml streptomycin.
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Extracted molecule |
total RNA |
Extraction protocol |
QIAshredder and RNeasy Mini Kit (Qiagen, UK) was used to isolate RNA from transfected U2OS cells according to the manufacturer’s protocol. Integrity of RNA was determined using a bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). A cDNA library including 5 replicates per condition was prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina Inc., San Diego, CA) according to the manufacturer´s recommendation. Briefly, 1µg of total RNA was used for first-strand synthesis performed with a random hexamer and SuperScript II (Life Technologies, Carlsbad, CA, USA). Second-strand synthesis was performed using dUTP and the Illumina-specific Second Strand Marking Master Mix. After end repair and A-tailing indexed adaptors were ligated to the cDNA fragments. Fragments successfully ligated with adaptor molecules on both ends were enriched by PCR for 15 cycles and purified with AMPure XP Beads (Beckman Coulter Inc., Brea, CA). The final libraries were quality checked on an Agilent Bioanalyzer and quantified with qPCR using a commercially available PhiX-library (Illumina Inc., San Diego, CA) as a reference. All samples were run in five biological replicates and a total of 30 equimolarily pooled samples were sequenced two High Output flow cells on an Illumina NextSeq in a paired end run with 2x 75 cycles.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
NextSeq 550 |
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Data processing |
Alignment: Star 2.6.1a_08-27 Transcript Counts: featureCounts (subread package) 1.4.4 Raw RNA-Seq reads were aligned to the human hg19 genome using STAR. considered differentially expressed. Gene read counts are generated using featureCounts and differential expression was analyzed using DESeq2. Genes with adjusted p-value lower than 0.05 and a log2 fold change of 1 or -1 were considered differentially expressed. Genome_build: hg19 Supplementary_files_format_and_content: read counts per gene
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Submission date |
Oct 16, 2019 |
Last update date |
Jun 01, 2020 |
Contact name |
Gesa Richter |
E-mail(s) |
gesa.richter@medunigraz.at
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Organization name |
Medical University Graz
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Street address |
Neue Stiftingtalstrasse 6
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City |
Graz |
ZIP/Postal code |
8010 |
Country |
Austria |
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Platform ID |
GPL21697 |
Series (1) |
GSE138938 |
β-catenin regulates FOXP2 transcriptional activity via multiple binding sites |
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Relations |
BioSample |
SAMN13039501 |
SRA |
SRX7004959 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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