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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 09, 2019 |
| Title |
RC-77T/E-CD133-plus-2 |
| Sample type |
SRA |
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| Source name |
African American-Prostate
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| Organism |
Homo sapiens |
| Characteristics |
ethnicity: African American
tissue: prostate
cell line: ADT resistant
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| Treatment protocol |
20.0 μmol/L of Biculatamde were added for 24 h, DMSO (control) to dishes. Media were changed after 48 h. Biculatamide treatment continued until the resistant clones were formed. Biculatamide resistant clones were treated with 20.0 μmol/L of Enzalutamide until the resistant Enzalutamide clones were formed. Cells were reseeded once each week supplemented with the appropriate treatments. Cells from control plates were maintained until resistant clones were cryopreserved to maintain a constant culture time for control and experimental lines.
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| Growth protocol |
RC-77-N, RC-77T/E and E006AA-hT were cultured in DMEM supplemented with 10 % FBS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using Qiagen RNeasy Plus Mini Kit (Qiagen). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity were checked with Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
RNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Human/Mouse/Rat probe) (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using a DNA Chip on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were multiplexed and clustered on one lane of a flowcell. After clustering, the flowcell were loaded on the Illumina HiSeq instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE). Image analysis and base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated from Illumina HiSeq will be converted into fastq files and de-multiplexed using Illumina bsl2fastq v. 2.17 program. One mis-match will be allowed for index sequence identification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument.
After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trim Sequences Module in CLC Genomics Workbench 10.0.1.
The trimmed reads were mapped separately to the long noncoding RNA and gene regions of the Homo sapiens GRCh38 reference genome available on ENSEMBL using the RNA-Seq Analysis Module in CLC Genomics Workbench 10.0.1. BAM files, Unique gene hit counts and Unique transcript hit counts for each mapping were generated as a result of this step.
After extraction of gene and transcript hit counts, the hit counts tables were used for downstream differential expression analysis. Using Kal’s test, a comparison of gene expression between the groups of samples was performed. The Kal’s test was used to generate p-values and Log2 fold changes. Genes and transcripts with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes and transcripts respectively for each comparison.
A gene ontology analysis was performed on the statistically significant set of genes by implementing the Hypergeometric tests in CLC Genomics Workbench 10.0.1. The goa human GO list was used to cluster the set of genes based on their biological process and determine their statistical significance
Genome_build: GRCh38 reference genome available on ENSEMBL
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| Submission date |
Jun 04, 2018 |
| Last update date |
Jul 09, 2019 |
| Contact name |
Mohammad Saleem |
| Organization name |
University of Minnesota
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| Department |
Urology
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| Lab |
Molecular Therapeutics and Cancer Health Disparity
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| Street address |
2231 6th St SE
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| City |
Minneapolis |
| State/province |
Mn |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE115307 |
Race-specific transcriptome and Long non-coding RNA of ADT-resistant African-American prostate cancer cell models. |
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| Relations |
| BioSample |
SAMN09340043 |
| SRA |
SRX4161264 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3175249_LncRNA-RC-77TE-CD133_plus-2_GE.xlsx |
1.4 Mb |
(ftp)(http) |
XLSX |
| GSM3175249_mRNA-RC-77TE-CD133-plus-2_GE.xlsx |
6.3 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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