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Status |
Public on Dec 07, 2020 |
Title |
BT20_shControl_24VC |
Sample type |
SRA |
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Source name |
BT20
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Organism |
Homo sapiens |
Characteristics |
ectopic wwox expression: shControl time of stimulation: 24h_control tissue: breast cancer cell line BT20
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Treatment protocol |
Lentiviral transduction with shRNA gene silencing system. Retroviral transfection for WWOX cDNA inserion.
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Growth protocol |
T47D and MCF7 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC), and grown according to the manufacturer’s protocol in RPMI-1640 Medium (Gibco) with 2 mM L-glutamine (Gibco), 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, 1500 mg/L sodium bicarbonate supplemented with 10% heat inactivated fetal bovine serum (Gibco), antibiotics (Gibco, 0.05 mg/ml penicillin; 0.05 mg/ml streptomycin; 0.1 mg/ml neomycin) and human insulin (ITS insulin/transferrin/selenous acid Premix, BD Biosciences), in a humidified atmosphere containing 5% CO2 at 37°C. BT20 breast carcinoma cell line was obtained from CLS (Cell Lines Service GmbH, Eppelheim, Germany) and grown according to the manufacturer’s protocol in DMEM: Ham’s F12 medium (1:1 mixture) supplemented with 2 mM L-glutamine (Gibco), 10% heat inactivated fetal bovine serum (Gibco) and antibiotics (Gibco, 0.05 mg/ml penicillin; 0.05 mg/ml streptomycin; 0.1 mg/ml neomycin) in a humidified atmosphere containing 5% CO2 at 37°C. Mammary gland adenocarcinoma cell line MDA-MB-231 was obtained from the American Type Culture Collection (ATCC) and was cultured in Advanced DMEM (Dulbecco’s modified Eagle’s medium) with 10% heat-inactivated fetal bovine serum, 4500 mg/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer and antibiotics (penicillin, 50 U/ml; streptomycin, 50 µg/ml; and neomycin, 100 µg/ml), in a humidified atmosphere of 5% CO2 at 37°C.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted with TRIzol reagent. The libraries for sequencing were prepared according to Piero Carnici’s protocol [Takahashi, et al., PMID:22362160]. Briefly, polyadenylated and non-poliadenylated RNA was reverse transcribed into cDNA with usage of random prime containing EcoP15I sequence. Next step was biotinylation of the cap site and the 3’ends of hybridized RNAs. Non-hybridized ssRNAs were digested and 5’ complete cDNAs hybridized to biotin-tagged, cap-containing RNAs were separated on streptavidin coated magnetic beads. cDNA was released from RNA and a double stranded 5’ linker with a barcode and EcoP15I sequences was ligated. Subsequently, using the biotinylated 2nd SOL primer, a second strand cDNA was synthetized. The digestion with EcoP15I allowed to cleave 27 bp fragments inside the 5’ end of the cDNA. In the 3’ end of cDNA a 3’ linker with 3’ Ilumina primer sequence was ligated. Next, constructed CAGE tags were amplified and purified. The prepared libraries were sequenced on Illumina HiSeq System.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
CAGE |
Instrument model |
NextSeq 550 |
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Data processing |
Processing and analysis of sequenced nAnT-iCAGE data was done basing on the workflow proposed by the Data Analysis Center of the Cell Innovation Program (https://cell-innovation.nig.ac.jp/maser/Applications/CAGE-seq_en.html), employing Galaxy – an open source, web-based platform. FASTQ sequenced data files were first assessed for proper quality by FastQC tool and splitted by a barcode sequence by means of Barcode Splitter tool. AfterQC tool was used for filtering, barcode trimming, error removal and quality check of fastq files. Burrows-Wheeler Alignment tool (BWA) was used for reads alignment and mapping to the human reference genome (GRCh38). The data were recorded in SAM (Sequence Alignment/Map) format, transformed to BAM (Binary Sequence Alignment Map) files and alignments sorted by means of SAMtools package. Identification of transcription start sites was performed with CAGEr (Haberle, et al., PMID:25653163). Briefly, tag counts were normalized to one million tags using simple TPM and transcription start sites (TSS) were clustered using paraclu. Finally, all genome mapped TSS were cross-identified using FANTOM5 human hg38 true-TSS library (http://fantom.gsc.riken.jp/5/datafiles/reprocessed/). All above done using CAGEr. Genome_build: GRCh38/hg38 Supplementary_files_format_and_content: Spreadsheet file of hg38 genome coordinates of identified TSS including TPM normalized signals of all samples and cross-identified according to FANTOM5 project: TSScoordinates_TPMexpression_all_samples_F5mapped.txt; Gene expression was counted as a summary of all TSS of a single gene. Spreadsheet file of gene expression of all samples: TPMexpression_all_genes_all_samples merged.txt
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Submission date |
Nov 14, 2019 |
Last update date |
Dec 08, 2020 |
Contact name |
Andrzej K. Bednarek |
E-mail(s) |
andrzej.bednarek@umed.lodz.pl
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Phone |
+48 604583839
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Organization name |
Medical University in Lodz
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Department |
Department of Molecular Carcinogenesis
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Street address |
Zeligowskiego St. 7/9
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City |
Lodz |
ZIP/Postal code |
90-752 |
Country |
Poland |
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Platform ID |
GPL21697 |
Series (1) |
GSE140406 |
WWOX gene and estrogen receptor interplay in breast cancer |
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Relations |
BioSample |
SAMN13284971 |
SRA |
SRX7141375 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4160705_BT20_shC_24VC.txt.gz |
481.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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