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| Status |
Public on Aug 05, 2023 |
| Title |
22Rv1NCOA6KO #4-91_untreated_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Prostate carcinoma epithelial cell line derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Prostate cancer cells
cell line: 22Rv1
genotype: NCOA6 knockout
treatment: Untreated
|
| Treatment protocol |
The 22Rv1Ctrl cell line was established by transfecting 22Rv1Par. cells with PX459 plasmids to transiently express Cas9. The 22Rv1NCOA6KO #2-74, #2-96, #4-91 and #4-105 cell lines were established by transfecting 22Rv1Par. cells with constructed PX459 plasmids to transiently co-express Cas9 and sgRNA targeting the exon 5 or 6 of the NCOA6 gene. The transfected cells were cultured in RPMI 1640 complete medium containing 5 µg/ml puromycin (P8833; Sigma) for 2 days. Selected cells were seeded into 150 mm dishes at a density of 200 cells per dish to grow clones. Individual clones were picked up and expanded. Positive clones including the 22Rv1NCOA6KO #2-74, #2-96, #4-91 and #4-105 cell lines were identified by PCR-based genotyping, DNA sequencing, and Western blot analysis.
|
| Growth protocol |
The 22Rv1-derived cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (10437028; Thermo Fisher) and 1% penicillin (10000 IU)/streptomycin (10000 µg/ml) solution (30-002-CI; Corning). Cells were maintained at 37°C in a 5% CO2 cell culture incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA samples were extracted from the 22Rv1-derived cell lines using the TRIzol reagent (15596018; Thermo Fisher) followed by purification with the RNeasy Mini Kit (74106; Qiagen) according to the manufacturer's instructions. Quality of the samples was checked by using the Nanodrop ND-1000 and Agilent Bioanalyzer Nano Chip.
|
| Label |
Cy3
|
| Label protocol |
50 ng of total RNA sample that had passed the quality check was mixed with Agilent's RNA Spike-Ins. The samples were amplified and labeled with Cy3 by using the Agilent Quick Amp Labeling Kit (Agilent p/n 5190-2331) according to the protocol (version 6.7). As a result, the Cy3-labeled cRNA was generated. The cRNA samples were purified using Qiagen RNeasy mini spin columns. Yield and dye incorporation of the samples were measured on Nanodrop.
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| |
|
| Hybridization protocol |
The samples were fragmented. 0.6 μg of the fragmented sample and hybridization mix was loaded onto each of the Agilent G3 Human Gene Expression v3 8x60K Expression arrays (G4851C). The slides were hybridized in Agilent Hybridization Chamber at 65°C at a rotation rate of 10 rpm for 17 hours. The hybridized slides were washed with the Agilent Expression Wash Buffer Set 1 and 2 as per the Agilent protocol.
|
| Scan protocol |
Once dry, the slides were scanned with the Agilent Scanner (G2565BA) using Scanner Version C and Scan Control software (version A.8.5.1). Data extraction and quality assessment of the microarray data were performed using Agilent Feature Extraction Software (Version 11.0.1.1).
|
| Description |
NCOA6 knockout
|
| Data processing |
The Bioconductor “limma” package (http://bioconductor.org) was used to analyze the microarray data. The data background was corrected using the “normexp” method with an offset of 16 added to the intensities. The background-corrected data were then log2-transformed and quantile-normalized.
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| |
|
| Submission date |
Aug 05, 2020 |
| Last update date |
Aug 05, 2023 |
| Contact name |
Yonghong Liu |
| E-mail(s) |
yonghongliu2017@gmail.com
|
| Phone |
7137984684
|
| Organization name |
Baylor College of Medicne
|
| Department |
Molecular & Cellular Biology
|
| Lab |
N630
|
| Street address |
One Baylor Plaza
|
| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL21185 |
| Series (2) |
| GSE155754 |
Identification of differentially expressed genes influenced by NCOA6 in 22Rv1 human prostate cancer cells |
| GSE155757 |
NCOA6 is a potent prostate cancer suppressor essential for NFY-mediated restriction of EGFR overexpression |
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