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| Status |
Public on May 02, 2019 |
| Title |
CFTRpos_Nontreated_4 |
| Sample type |
SRA |
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| Source name |
Caco2 cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Colonic adenocarcinoma-derived cell line
genotype: NonCF
clone: Y5
treatment: none
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| Treatment protocol |
Cells were exposed to 100ng/ml TNFa for 2 hours prior to harvesting RNA for RT PCR and RNAseq
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| Growth protocol |
Caco-2 cells obtained from the American Type Culture Collection (ATCC) were maintained in Eagle’s minimum essential medium with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. Cells were cultured at 37°C in an atmosphere of 95% O2 - 5% CO2
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested using RNAeasy kit per manufacturer instructions, Qiagen Inc, Valencia, CA)
RNA-Seq libraries were generated using TruSeq stranded Total RNA Ribo-Zero human gold kit (Illumina, San Diego, CA) and the quality of resulting libraries was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). Sequencing was carried out using either an Illumina HiSeq 2500 Rapid Run flowcell – 2x100bp run or an Illumina NextSeq 550 HO - 2x75bp run, as indicated below.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
Generated using CRISPR/Cas9-mediated mutagenesis of CFTR
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| Data processing |
Trimming and filtering of the reads to remove adaptor sequences and low-quality nucleotides were performed using Trimmomatic (v 0.36). 100bp samples were trimmed to 75bp before continuing with the rest of the analysis.
The filtered reads were aligned to the UCSC human genome hg19 as a reference using Bowtie2 (v 2.3.4.3) and Tophat (v 2.1.1). Assembly of transcriptomes and quantification of their expression was performed using Cufflinks (v 2.2.1). Expression levels were expressed as fragments per kilobase of exon per million fragments mapped (FPKM).
Statistical significance in differential gene expression between groups was determined with Cuffdiff (v 2.2.1). Both p and q values were determined and defined as uncorrected p-value of the test statistic and FDR-adjusted p-value of test-statistic, respectively.
Data were further analyzed and visualized using CummeRbund run under the R package (v 3.4.4)
Hierarchical clustering (heat map) of genes with differential expression was generated using Complete Linkage as the clustering method, Euclidian distance as the similarity measure, and Z-Score as normalization with TIBCO Spotfire Software (Palo Alto, CA). Pathway analysis was done using Gene Set Enrichment Analysis (GSEA) (Broad Institute, software.broadinstitute.org/gsea), a computational method that determines statistical significance of a priori defined set of genes between two groups. The Hallmark gene set from the Molecular Signature Database (MSigDB) collection was used to explore overrepresented pathways. It comprises of 50 hallmarks condensed from over 4,000 overlapping gene sets from v4.0 MSigDB collections C1 through C6.
Genome_build: hg19
Supplementary_files_format_and_content: Excel file for each sample including FPKM values and locus
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| Submission date |
Apr 23, 2019 |
| Last update date |
May 04, 2019 |
| Contact name |
Aura Perez |
| E-mail(s) |
[email protected]
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| Phone |
216-368-6894
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| Organization name |
CWRU
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| Department |
Pediatrics
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| Street address |
10900 Euclid Avenue
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| City |
Cleveland |
| State/province |
OH |
| ZIP/Postal code |
44106-4948 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE130226 |
Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks |
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| Relations |
| BioSample |
SAMN11487409 |
| SRA |
SRX5727054 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3734147_Caco2_WT4_Unt_S8.xlsx |
1.1 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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