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| Status |
Public on Jun 04, 2018 |
| Title |
LY2-SRC1-shRNA-n-4A |
| Sample type |
SRA |
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| Source name |
LY2 Breast Cancer Cell Line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LY2
cell type: endocrine-resistant breast cancer
treatment: Tamoxifen
shRNA: shSRC1 (SRC1 targeted shRNA)
replicate number: Replicate 4
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| Treatment protocol |
Prior to treatment, cells were steroid depleted for 72 hours, followed by 4-OHT [ 10^-7 M] treatment for 8 hours prior to RNA-sequencing.
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| Growth protocol |
Endocrine resistant LY2 breast cancer cells were maintained in phenol red free Minimum Essential Medium Eagle (PRF-MEM, Sigma, Darmstadt, Germany) supplemented with 10 % charcoal dextran stripped fetal calf serum (CDS-FCS, Sigma), 1 % L-glutamine (LG, Sigma), 1 % penicillin-streptomycin (PS, Sigma) and 4-hydroxytamoxifen (4-OHT, Sigma) 10-8 mol/L. LY2 shSRC-1 and LY2 shNT cells were maintained in LY2 cell culture media containing puromycin (500 ng/mL, Sigma) to maintain stable knockdown expression.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using standard BGI extraction protocols. RNA was extracted with average concentrations of 500 ng/µl determined by QubitFluorometer, Agilent 2100 bioanalyser, and nanodrop. BGI analysed the RNA integrity using the Agilent 2100 bioanalyser achieving a >9.5 RNA integrity number for all samples. Total quantity of RNA was above the cut off of ≥ 5µg
LIbrary construction was carried out using Illumina®TruSeq RNA Sample Prep Kit. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The RNA-seq alignment was carried out using Tophat2 (version 2.0.12) to Ensembl's GrCH37 version 75 transcriptome assembly
RNA-seq counts were calculated using HTSeq (version 0.6.1) and uploaded into R
The HTSeq counts were processed by DESeq2 and were assessed for differential expression
Genome_build: Ensembl GrCH37 version 75 transcriptome
Supplementary_files_format_and_content: DESeq normalised counts (counts(dds, normalized=T)
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| Submission date |
Jun 04, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Leonie Young |
| Organization name |
Royal College of Surgeons, Ireland
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| Department |
Surgery
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| Lab |
Endocrine Oncology Research Group
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| Street address |
York Street
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| City |
Dublin |
| ZIP/Postal code |
Dublin 2 |
| Country |
Ireland |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE99647 |
RNA-sequencing of shSRC-1 and shNT tamoxifen treated LY2 cells |
| GSE99649 |
RNA-sequencing and MeDIP-sequencing of shSRC-1 and shNT tamoxifen treated LY2 cells |
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| Relations |
| BioSample |
SAMN07192383 |
| SRA |
SRX2882372 |
| 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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