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Sample GSM2639667 Query DataSets for GSM2639667
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
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
BioSample SAMN07163020
SRA SRX2850861

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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