|
| Status |
Public on May 24, 2018 |
| Title |
HCT-116 cells untransfected |
| Sample type |
SRA |
| |
|
| Source name |
APC wild-type, CTNNB1ΔSer45
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT-116
cell type: colon carcinoma
treatment: transfected with reagent only (mock)
|
| Treatment protocol |
1.0 x 105 HCT-116 cells were seeded in each well of a 6-well plate, 16-20 hours before transfection. Transfections were performed on consecutive days either using no siRNA (mock transfection) or using pooled siRNA (Dharmacon) with scrambled sequence (non-targeting pool, D-001810-10-05), or targeting APC (L-003869-00-0005). Transfections followed manufacturer's instructions for the transfection reagent (Dharmafect 2, T-2002-01) and used a combination of 4 μL of Dharmafect 2 and 5 μL of 10 μM siRNA stock solution. Media changes were performed 6 hours after each transfection, and cell harvest was performed 48 hours after the second transfection.
|
| Growth protocol |
HCT-116 cells were purchased from the American Type Culture Collection (ATCC) and cultured in McCoy's 5A medium (GIBCO catalog #16600082) supplemented with 10% fetal bovine serum. Cells had been cultured for fewer than 20 passages at the time of the experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by washing with Dulbecco’s PBS, addition of 500 μL Trizol (Life Technologies catalog # 15596-026), and scraping. 50 μg glycogen (RNase-free) was mixed into each sample. A standard Trizol isolation protocol was then performed, with final resuspension in nuclease-free water.
Three single-read libraries were prepared using the TruSeq RNA Library Preparation Kit (Illumina catalog # RS-122-2001) according to Illumina's instructions. Agilent Bioanlyzer was used to quantify libraries, and the OSU Genomics Shared Resource performed next-generation sequencing on an Illumina HiSeq instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
mRNA-seq
|
| Data processing |
Initial data processing was performed by The Ohio State University Biomedical Informatics Shared Resource to generate .qseq files from base call files.
Files in .qseq format were sorted and filtered to generate sorted and export text files that could be analyzed using the CuffLinks software program.
The CuffLinks program was used to assign reads to transcripts, perform quantification and calculate statistical significance. The output of the Cuffdiff program included transcript levels expressed in FPKM units and both p-values and (FDR-adjusted) q-values based on fold-changes. The threshold for statistically significant differences between the anti-APC siRNA condition and either the scrambled siRNA or mock-transfected conditions was established as q-values less than 0.05.
Genome_build: hg19
Supplementary_files_format_and_content: The files RNA-seq_RPKM_HCT-116_siAPC_vs_untreated_cuffDiff.txt and RNA-seq_RPKM_HCT-116_siAPC_vs_siScrambled_cuffDiff.txt are text files that each contain transcript abundance data expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for two conditions. The first compares transcript abundance in untreated vs. anti-APC siRNA-transfected HCT-116 cells, while the second compared transcript abundance in scrambled siRNA- vs. anti-APC siRNA-transfected cells. Files were generated using the Cuffdiff program, and p- and q-values were calculated from fold-changes in order to estimate statistical significance.
Supplementary_files_format_and_content: In the file "RNA-seq_RPKM_HCT-116_siAPC_vs_siScrambled_cuffDiff.txt", q1 refers to the "siAPC" sample from column H, and q2 refers to the "siScrambled" or "siSCR" sample from column I.
Supplementary_files_format_and_content: In the file "RNA-seq_RPKM_HCT-116_siAPC_vs_untreated_cuffDiff.txt", q1 refers to the "siAPC" sample from column H, and q2 refers to the "untreated" or "none" sample from column I.
|
| |
|
| Submission date |
May 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Joanna Groden |
| E-mail(s) |
groden.2@osu.edu
|
| Phone |
(614) 688-4301
|
| Organization name |
The Ohio State University, College of Medicine
|
| Department |
Department of Cancer Biology and Genetics
|
| Street address |
986 BRT 460 West 12th Ave.
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43210 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE99263 |
Gene expression data from the HCT-116 colon cancer cell line in the presence or absence of siRNA targeting APC |
| GSE99264 |
Chromatin binding profiles of the APC tumor suppressor and changes in gene expression following APC silencing |
|
| Relations |
| BioSample |
SAMN07163020 |
| SRA |
SRX2850861 |
