|
| Status |
Public on Sep 15, 2015 |
| Title |
shCTRL infected 7 hrs 1 |
| Sample type |
SRA |
| |
|
| Source name |
HT-29 cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: colon
cell type: epithelial
disease: colon adenocarcinoma
|
| Treatment protocol |
Cells were grown to confluency and remained confluent for two days. They were then treated for 48 hours with IPTG to induce control or CAP-D3 shRNA expression. Cells were infected with Salmonella enterica serovar Typhimurium SL1344 in RPMI +10% FBS at a multiplicity of infection (MOI) of 10.
|
| Growth protocol |
HT-29 cells were cultured in RPMI 1640 media with L-glutamine, 10% fetal bovine serum (Gibco), and 1% penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol (Invitrogen) was used to harvest total RNA from tissues and cells according to manufacturer's protocol. Samples were DNase treated and run on RNAeasy columns (Qiagen).
Directional, cDNA libraries made from cellular mRNAs were generated from the other 16 samples using the Illumina TruSeq RNA library kit and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2500, according to standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ML34
|
| Data processing |
Samples were evaluated for quality and trimmed of adapters and low-quality bases using Trim Galore! v.0.3.3 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with default settings to produce FastQC plots and trimmed read files.
Trimmed reads were then aligned and quantified using TopHat (v.2.0.8) and Cufflinks (v2.2.0). The Cufflinks work flow was followed using default settings but with Tophat set to "sensitive" mode (--b2-sensitive) with inner mate pair distance and standard deviation set to 100 and 30, respectively (-r 100 --mate-std-dev 30), based on library parameters.
Cuffdiff was used to assess differential expression. An adjusted P-value < 0.05 and a fold change >1.75 was deemed significant
Genome_build: hg19
Supplementary_files_format_and_content: Supplementary Table 2. Analysis of differentially expressed genes in uninfected control shRNA and CAP-D3 shRNA expressing HT-29 cells. Total mRNAs from 3 different experiments per cell line were analyzed (control shRNA experiments c_0, c_1, c_3 and CAP-D3 shRNA experiments t_0, t_1, t_3). Normalized FPKM values (columns O-T) were averaged and used to calculate the log base 2 fold change from CAP-D3 shRNA expressing cells to control shRNA expressing cells (column J). Adjusted p-values (q-value) are shown in column M. Genes significantly downregulated in CAP-D3 shRNA expressing cells are highlighted in blue while genes significantly upregulated in CAP-D3 shRNA expressing cells are highlighted in red. Genes were considered significant if the adjusted p-value was <0.05 and the log fold change was at least 1.75 fold.
Supplementary_files_format_and_content: Supplementary Table 3. Analysis of genes that are differentially expressed in control shRNA and CAP-D3 shRNA expressing HT-29 cells as a result of infection. Cells were infected with S. typhimurium and transcript levels were analyzed at 0.5 hours (columns E-L) and 7 hours post infection (columns M-T). Total mRNAs from 2 different experiments per cell line were analyzed for the 0.5 hour timepoint and from 3 different experiments for the 7 hour experiment. Normalized FPKM values were averaged and used to calculate the log base 2 fold change from uninfected to 0.5 hour infected control shRNA (sheet 1) and CAP-D3 shRNA (sheet 2) expressing cells (column H). Adjusted p-values (q-value) are shown in column K. Genes significantly downregulated at the 0.5 hour post-infection timepoint are highlighted in blue while genes significantly upregulated at the 0.5 hour post-infection timepoint are highlighted in red. Genes were considered significant if the adjusted p-value was <0.05 and the log fold change was at least 1.75 fold. There are two spreadsheets; the first contains the differentially expressed genes for the control shRNA expressing cells and the second contains the genes for the CAP-D3 shRNA expressing cells.
|
| |
|
| Submission date |
Oct 20, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Michelle Suzanne Longworth |
| E-mail(s) |
longwom@ccf.org
|
| Phone |
216-618-6114
|
| Organization name |
Cleveland Clinic Foundation
|
| Department |
Molecular Genetics
|
| Lab |
Longworth
|
| Street address |
9500 Euclid Ave NE20
|
| City |
Cleveland |
| State/province |
OH |
| ZIP/Postal code |
44195 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE62520 |
RNA-sequencing of mRNAs from control and CAP-D3 deficient Salmonella infected HT-29 cells |
|
| Relations |
| BioSample |
SAMN03120862 |
| SRA |
SRX736474 |
