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Sample GSM4160705 Query DataSets for GSM4160705
Status Public on Dec 07, 2020
Title BT20_shControl_24VC
Sample type SRA
 
Source name BT20
Organism Homo sapiens
Characteristics ectopic wwox expression: shControl
time of stimulation: 24h_control
tissue: breast cancer cell line BT20
Treatment protocol Lentiviral transduction with shRNA gene silencing system. Retroviral transfection for WWOX cDNA inserion.
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection CAGE
Instrument model NextSeq 550
 
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
 
Submission date Nov 14, 2019
Last update date Dec 08, 2020
Contact name Andrzej K. Bednarek
E-mail(s) andrzej.bednarek@umed.lodz.pl
Phone +48 604583839
Organization name Medical University in Lodz
Department Department of Molecular Carcinogenesis
Street address Zeligowskiego St. 7/9
City Lodz
ZIP/Postal code 90-752
Country Poland
 
Platform ID GPL21697
Series (1)
GSE140406 WWOX gene and estrogen receptor interplay in breast cancer
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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