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| Status |
Public on May 20, 2013 |
| Title |
AX4 grown on Sa, replicate 1 |
| Sample type |
SRA |
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| Source name |
AX4 grown on Sa
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| Organism |
Dictyostelium discoideum |
| Characteristics |
strain: AX4
growth on bacteria: Staphylococcus aureus
replicate: 1
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| Treatment protocol |
1-3x10^7 D. discoideum cells were mixed with 2 ml of thick bacterial culture and spread evenly on a 10-cm petri plate of buffered agar. After 14-16 hours, amoebae and bacteria were harvested and resuspended in ice-cold mSor buffer to wash away bacteria by low centrifugation (200xg at 4oC). Supernatant with bacteria was discarded. This procedure was repeated 2-4 times until no visible bacteria were detected in supernatant.
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| Growth protocol |
D. discoideum was recovered on K. pneumoniae bacterial lawn on SM medium, then grown axenically on HL5 medium and maintained at mid-log phase (2.5x10^6 cells/ml) before subsequent experiments.
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| Extracted molecule |
total RNA |
| Extraction protocol |
2.5x10^7 washed amoebae cells were immediately lysed in TRIZOL® reagent, and total RNA was prepared according to the manufacturer's protocol (Invitrogen by Life Technologies).
We processed 20 μg of total RNA through one round of poly-A selection, RNA fragmentation, first-strand and second-strand cDNA synthesis. Two biological replicates were analyzed for each condition. We sequenced the cDNA libraries (read length = 35 bases) on a high-throughput Illumina Genome Analyzer II using the manufacturer's recommended pipeline (versions 1.2 and 1.3).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
AX4_Sa_bio1
mRNA
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| Data processing |
The data was mapped to the D. discoideum genome with bowtie version 0.12.7. Read qualities were used. Other parameters: seed length = 28, allowed mismatches = 2, allowed up to 1 alignment per read, with bowtie options -a, --strata, --best. Unmapped reads were trimmed from 3' by 2 nucleotides, which was repeated 5 times.
Raw expression values were computed as the number of reads that were uniquely mapped to the exons of genes.
Normalized expression were scaled by mappable exon lengths. This normalization is similar to the RPKM normalization (Reads Per Kilobase of exon per Megabase of library size), but instead of dividing by exon lengths we used the length of uniquely mappable parts of the exon. To obtain uniquely mappable parts, all possible subsequences of the reference genome (of the same length as reads in raw data) are mapped back to the reference genome, obtaining the number of uniquely mapped sequences to the exons (Exon_mappable). As a library size we used the total number of all uniquely mapped reads from the experiment, excluding the non-polyadenylated genes (N_unique). Normalized expressions were computed as follows: Exp = 10^9*raw/(N_unique * Exon_mappable).
D. discoideum Chromosome: 1,2,3,4,5,6,M, and floating contigs (created: 05-13-2009 13:53) from the DictyBase (chromosomes 1,2,3,4,5,6 and mitochondrial DNA are the same as on NCBI assembly "dicty_2.7"). Regions [3016085, 3768655] from chr2, [64985, 72996] from chrBF and [42801, 78150] from chrR were masked.
Genome_build: dicty_2.7
Supplementary_files_format_and_content: Processed data files are tab-separated files with two columns: the first containing gene names and the second its expression. Files ending with "_expr_raw.tab" contain raw expression values, while files ending with "_expr_norm.tab" contain normalized expression values.
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| Submission date |
Apr 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Gad Shaulsky |
| E-mail(s) |
gadi@bcm.edu
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| Organization name |
Baylor College of Medicine
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| Department |
Molecular and Human Genetics
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| Street address |
One Baylor Plaza
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL9379 |
| Series (1) |
| GSE46386 |
Bacterial discrimination by Dictyostelid amoebae reveals the complexity of ancient interspecies interactions |
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| Relations |
| BioSample |
SAMN02056344 |
| SRA |
SRX271995 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1129603_AX4_Sa_expr_norm1.tab.gz |
111.1 Kb |
(ftp)(http) |
TAB |
| GSM1129603_AX4_Sa_expr_raw1.tab.gz |
48.1 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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