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| Status |
Public on Apr 15, 2014 |
| Title |
NLS 1h developing; replicate 1 |
| Sample type |
SRA |
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| Source name |
NLS 1h developing
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| Organism |
Dictyostelium discoideum |
| Characteristics |
strain: AX2
genotype: NLSex-GFP-GtaC/gtaC
replicate: 1
developmental time: 1 h
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| Treatment protocol |
For development on non-nutrient agar, cells were washed with developmental buffer (DB) and plated on solidified DB agar at a density of 5.2^105 cells/cm2
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| Growth protocol |
Wild-type and gene deletion cell lines were cultured axenically in HL5 supplemented with antibiotics.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At each time point, we harvested the cells directly into 1 mL of Trizol® (life technologies, CA, USA) and extracted total RNA according to the manufacturer’s recommended protocol. We performed two rounds of poly-A selection and fragmented 100 ng of the resulting mRNA into approximately 200 bases fragments. We prepared cDNA and the second strand.
We prepared Illumina multiplexed libraries. We prepared 15 (or 20) individual libraries separately (one from each sample) and added a unique barcode to each library at the final step of PCR amplification. We pooled equal amounts of DNA from each library and sequenced one pool per lane of a flow cell on Illumina Genome Analyzer II using the manufacturer's recommended pipeline (versions 1.2 and 1.3, read length = 50 bases)
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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 |
NLS_1h_bio1
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| Data processing |
After demultiplexing, 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 gene exons.
Normalized expression were scaled with 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 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).
Genome_build: D. discoideum Chromosomal DNA: 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.
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 "_raw.tab" contain raw expression values while files ending with "_nor.tab" contain normalized expression values.
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| Submission date |
Feb 11, 2014 |
| 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) |
| GSE54866 |
Nucleocytoplasmic shuttling of a GATA transcription factor functions as a development timer |
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| Relations |
| BioSample |
SAMN02640014 |
| SRA |
SRX469376 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1325355_NLS_1hr_bio1_nor.tab.gz |
110.5 Kb |
(ftp)(http) |
TAB |
| GSM1325355_NLS_1hr_bio1_raw.tab.gz |
48.9 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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