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Status |
Public on Oct 28, 2022 |
Title |
LNCaP cells, DMSO treatment |
Sample type |
SRA |
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Source name |
prostate
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Organism |
Homo sapiens |
Characteristics |
tissue: prostate cell line: LNCaP cell type: prostate adenocarcinoma cells genotype: WT treatment: DMSO (negative control)
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Treatment protocol |
Cells were seeded at 450,000 cells per 15 cm culture dish and allowed to attach to the dish overnight. The following day, enzalutamide was added at 5 μM concentration diluted from 100 mM stock in DMSO. Dilutions were prepared such that DMSO content in culture plates, including DMSO control, was always 0.1%. Enzalutamide treatment was performed for 4, 7, or 14 days at 5 μM treatment. Media was refreshed and enzalutamide was re-applied to cell culture dishes every 72 h. DMSO cells were seeded at the same density and treated at same schedule as enzalutamide treatment groups but harvested at 70-80% confluency.
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Growth protocol |
LNCaP cell line was purchased from ATCC. Cells were stored as frozen stock in vapor phase of LN2 and thawed prior to use. Cell lines were cultured 3 passages after thawing prior to experimentation and maintained for no longer than 30 total passages. LNCaP cells were cultured in RPMI 1640 (Corning), and All media were supplemented with 10% FBS (Atlanta Biologicals), 1 mM sodium pyruvate (Corning), penicillin (100 units/mL) and streptomycin (100 μg/mL) combination (Gibco), and 2 mM/L L-glutamine (Corning).
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Extracted molecule |
total RNA |
Extraction protocol |
The 15 cm cell culture dishes were removed from the 37 °C incubator, medium was removed via aspiration, and cells were immediately washed once with 10 mL of 37 °C phosphate buffered saline (PBS), pH 7.4. PBS was removed via aspiration and 3 mL 0.25% trypsin (Corning) was added to the dishes. Dishes were rotated for complete coverage of the trypsin and incubated on the benchtop for 30 seconds. Roughly 2 mL of the trypsin was removed via aspiration, and dishes were transferred to a 37 °C incubator for exactly 4 minutes. 10 mL of 4 °C RPMI supplemented with 10% FBS was used for resuspension of the trypsinized cell suspension. Viability was checked on Countess II FL automated cell counter to ensure >90% viability. Cells were then centrifuged 4 minutes at 400x g at 4 °C to pellet the cells. Supernatant was aspirated and discarded, then cells were washed with 1 mL 4 °C complete RPMI three times. Final resuspension of cells in 400 μL 4 °C complete RPMI was performed, and cell homogeneity and viability were checked to ensure singlet cells at >90% viability would be delivered to Indiana University School of Medicine Chemical Genomics Facility for 10X library preparation. The single cell gene expression analysis was conducted using a 10X Chromium single cell system (10X Genomics, Inc) and a NovaSeq 6000 sequencer (Illumina, Inc). To maximize quality of cell suspension, cell harvest was optimized to retain >95% viability at time of harvest following three washes in RPMI/10%FBS. Following storage on ice for delivery of cells to 10X sequencing facility, the single cell suspension was subsequently washed a fourth time with RPMI/10% FBS, recentrifuged, and cell debris, dead cells and cell aggregates were removed by discarding supernatant and resuspending cells. Each clean single cell suspension was then be counted with hemocytometer under microscope for cell number and cell viability. The single cell suspension viability was over >90% and minimal cell debris and aggregation were ensured prior to loading. A target of 10,000 cells was used for loading; in practice, following final analysis, between 6,890 and 11,620 individual cells were identified per sample after all processing steps were completed. The cells were applied to a single cell master mix with lysis buffer and reverse transcription reagents, following the Chromium NextGEM Single Cell 3’ Reagent Kits User Guide, CG000204 Rev D (10X Genomics, Inc). Along with the single cell gel beads and partitioning oil, the single cell master mixture containing the single cell suspension was dispensed onto a Single Cell Chip G in separate wells, and the chip loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library was be examined by Bioanalyzer and Qubit. The resulting library was sequenced in a custom program for 28b plus 91b paired-end sequencing on Illumina NovaSeq 6000. Depending on targeted cell recovery, roughly 33,000 – 82,000 reads per cell were generated and 91% of the sequencing reads reached Q30 (99.9% base call accuracy). A Phred quality score (Q score) was used to measure the quality of sequencing.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
CellRanger 6.0.1 (http://support.10xgenomics.com/) was utilized to process the raw sequence data generated.The FASTQ files were then aligned to the human reference genome hg38 with RNAseq aligner STAR [PMID: 23104886]. The aligned reads were traced back to individual cells and the gene expression level of individual genes were quantified based on the number of UMIs (unique molecular indices) detected in each cell. The filtered gene-cell barcode matrices generated by CellRanger were used for further analysis. Seurat was used for data normalization, data scaling, graph-based clustering, cluster marker genes idenfification. The CCA function from Seurat was used for integrative analysis of the samples. Assembly: hg38 Supplementary files format and content: Cell Ranger output files (barcodes.tsv, features.tsv, matrix.mtx)
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Submission date |
Oct 17, 2022 |
Last update date |
Oct 28, 2022 |
Contact name |
Changdeng Hu |
Organization name |
Purdue University
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Department |
Dept. of Medicinal Chemistry and Molecular Pharmacology
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Street address |
575 Stadium Mall Drive
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City |
West Lafayette |
State/province |
IN |
ZIP/Postal code |
47907 |
Country |
USA |
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Platform ID |
GPL24676 |
Series (2) |
GSE215943 |
Reprogramming Landscape Highlighted by Dynamic Transcriptomes in Therapy-induced Neuroendocrine Differentiation [scRNA-seq] |
GSE215945 |
Reprogramming Landscape Highlighted by Dynamic Transcriptomes in Therapy-induced Neuroendocrine Differentiation |
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Relations |
BioSample |
SAMN31322972 |
SRA |
SRX17919309 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6647951_DMSO_barcodes.tsv.gz |
52.8 Kb |
(ftp)(http) |
TSV |
GSM6647951_DMSO_features.tsv.gz |
325.6 Kb |
(ftp)(http) |
TSV |
GSM6647951_DMSO_matrix.mtx.gz |
169.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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