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| Status |
Public on May 14, 2024 |
| Title |
RC77N/E 10nM 1a25(OH)2D3 treated Rep 2 |
| Sample type |
SRA |
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| Source name |
RC77N/E
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| Organism |
Homo sapiens |
| Characteristics |
cell line: RC77N/E
cell type: non malignant prostate cell line
treatment: 10nM 1a,25(OH)2D3 treated
time: 24 hours
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| Treatment protocol |
For treatment, 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3] (Tocris Bioscience, Bristol, U.K., & Cat: 25-515-0U) reconstituted in 100% ethanol was used. Cells were then treated with 10 nM (physiological concentration) for 24 h[M1][JJ2]. Vehicle (ethanol) was included for every untreated control. The final ethanol percentage for treated and untreated (vehicle control) cell lines was 0.01%.
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| Growth protocol |
RC-77N/E was grown in a humidified incubator with 5% CO2 at 37 °C. In accordance with recent guidelines by the National Institutes of Health (NIH) on the authentication of key biological resources, cells were authenticated utilizing Short Tandem Repeat (STR) profiling against the American Type Culture Collection (ATCC) STR database (ATCC, Cat: ATCC 135-XV). RC-77N/E cells were routinely tested for mycoplasma contamination using MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, U.S.A., Cat: CUL001B). RC-77N/E cell line was provided by C.A. Casiano’s laboratory with permission from J.S. Rhim. RC-77N/E was grown in collagen-coated treated culture dishes in keratinocyte serum-free medium (K-SFM) supplemented with bovine pituitary extract and recombinant epidermal growth factor (Gibco, Grand Island, U.S.A., Cat:17-005-042) and was used for growing and maintaining the cells. Normocin 0.2% (Invivogen, San Diego, U.S.A., Cat: ANT-NR-1) was added to the medium for all cell lines to prevent contamination by mycoplasma, bacteria, or fungi.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Before handling RNA, surfaces and instruments underwent treatment with RNase Zap (Ambion AM9780, Austin, U.S.A.). RNA extraction from cultured cells followed the Rneasy Mini protocol (Qiagen 7414, Hilden, Germany). Initially, cells were rinsed with PBS, then lysed with buffer RLT and 10 μL/mL β-mercaptoethanol, utilizing 350 μL per T25 flask or 600 μL per 10 cm plate. Subsequently, the Rneasy Mini protocol, inclusive of on-column incubation with Rnase-free Dnase (Qiagen, Cat: 79254), was adhered to. Post washes, RNA was eluted using 30 μL Rnase-free water, and its concentration was determined employing a Nanodrop spectrophotometer. RNA samples were stored at −80 °C, with each sample being isolated and utilized individually in triplicate.
RNA sequencing was performed by the City of Hope Integrative Genomics Core facility using RNA sequencing libraries prepared with Kapa RNA mRNA HyperPrep kit (Kapa Biosystems, Wilmington, U.S.A., Cat KR1352) according to the manufacturer’s protocol. The final libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit and quantified with Qubit. Sequencing was performed on Illumina HiSeq 2500 with the single read mode of 51 cycles. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
VDRS-11
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| Data processing |
Sequencing was performed on Illumina HiSeq 2500 with the single read mode of 51cycle. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis.
Quality of the raw RNAseq read data was assessed using FastQC (v0.11.7)
Read trimming and adapter removal was performed using Trimmomatic (v0.36)
Trimmed reads were aligned to the reference genome (GrCh37) using HISAT2 (v2.1.0)
Gene expression was quantified from alignments using Stringtie (v1.3) and reported in transcripts per million (TPM)
Assembly: GrCh37
Supplementary files format and content: tab-delimited text matrix include TPM values for each Sample
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| Submission date |
May 14, 2024 |
| Last update date |
May 14, 2024 |
| Contact name |
Yate-Ching Yuan |
| E-mail(s) |
yyuan@coh.org
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| Organization name |
City of Hope
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| Street address |
1500 East Duarte
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| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE267396 |
1,25-Dihydroxyvitamin D3 Suppresses Prognostic Survival Biomarkers Associated with Cell Cycle and Actin Organization in a Non-Malignant African American Prostate Cell Line |
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| Relations |
| BioSample |
SAMN41391745 |
| SRA |
SRX24545234 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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