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| Status |
Public on Apr 01, 2017 |
| Title |
Mock4 |
| Sample type |
SRA |
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| Source name |
intestinal epithelial cell
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| Organism |
Rattus norvegicus |
| Characteristics |
host cell line: ATCC IEC-18
passage: 15-20
infection: IEC-18 with uninfected cell lysate
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each sample at 8 hour post infection using TRIzol reagent
RNA libraries were prepared for sequencing using standard Illumina protocols by Stanford University Functional Genomic Facility
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Barcode: GTCCGC
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| Data processing |
Raw reads were uploaded onto the CLC Genomics Workbench 8.0 (Qiagen) platform for independent alignments against the genomes of Rattus norvegicus (Ensembl.org/Rnor.6.0) and Toxoplasma Type II Me49 strain (ToxoDB-24, Me49 genome). All parameters were left at their default values.
Many genes are so highly conserved across evolution that they have sequences that are almost identical between Toxoplasma and rat. This makes it difficult to know exactly which reads in a given sample from infected cells derive from the Toxoplasma vs. rat versions of the gene. Because of this, we needed to identify and exclude such genes from our analysis. To do this, we first searched the uninfected and mock-infected RNASeq data for reads mapping to the Toxoplasma genome; because these samples were uninfected, any such reads would indicate spurious matches. We then compared the number of such reads to the number for the same gene in the infected samples where Toxoplasma infection is present. After normalizing total read numbers to be the same for each sample, any Toxoplasma gene that in the uninfected controls had ≥20% of the number of reads in the TZ or SPZ samples was deemed compromised and so it was excluded from all downstream analyses. Toxoplasma genes that had an average number of reads in the uninfected samples <20% of the average adjusted reads in the infected sample were left in the analysis but the read numbers from the infected sample were adjusted by subtracting the average number of reads in the uninfected and mock samples, after normalization for total read number. Genes with less than 5 exon reads mapping to the rat genome or to the parasite genome in all samples were excluded from further analysis. The number of total reads mapped to each genome after the adjustments described above was used to determine the RPKM (Reads Per Kilobase of transcript per Million mapped reads), rounded to the nearest one-tenth value, as the relative expression for each rat and Toxoplasma gene in each sample
SAMseq package for the R platform was used to identify genes with significant changes between two samples. To identify genes with statistically different expressions between samples, we set the delta (Δ) value at 10% FDR (False Discovery Rate) with q-value less than 5%.
Only genes with q-value less than 5% and an average of at least 5 exon reads in one of the two conditions being compared were considered for further analysis. Among these genes, only those with RPKM ratios ≥1.5 for the two samples being compared and consistent in the two infections with tachyzoites (“TZ” and “TZ+fzSPZ”) were included in the list of differentially expressed genes.
Genome_build: ToxoDB-24, Me49 genome
Genome_build: Ensembl.org/Rnor.6.0
Supplementary_files_format_and_content: tab delimited text files include gene ID, exon length, total gene reads and total exon reads obtained from CLC genomics for each sample when mapped to either Toxoplasma or Rat genome
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| Submission date |
Feb 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Pascale S Guiton |
| E-mail(s) |
[email protected]
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| Organization name |
Stanford University
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| Department |
Microbiology and Immunology
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| Lab |
Dr. John Boothroyd
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| Street address |
Sherman Fairchild Science Building Stanford School of Medicine 299 campus Drive
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305-5124 |
| Country |
USA |
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| Platform ID |
GPL20084 |
| Series (1) |
| GSE94473 |
An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells |
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| Relations |
| BioSample |
SAMN06294164 |
| SRA |
SRX2536755 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2476202_Mock4_RAT.txt.gz |
223.5 Kb |
(ftp)(http) |
TXT |
| GSM2476202_Mock4_TOXO.txt.gz |
46.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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