 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 14, 2013 |
| Title |
wildtype 18-hr replicate=2 |
| Sample type |
SRA |
| |
|
| Source name |
wildtype
|
| Organism |
Dictyostelium discoideum AX4 |
| Characteristics |
genotype: wildtype
replicate: 2
developmental stage: 18-hour
|
| Treatment protocol |
We developed 5×107 cells on a 5 cm nitrocellulose membrane for the number of hours specified in the text.
|
| Growth protocol |
We grew all the strains in liquid suspension in HL5 medium supplemented with streptomycin (50 mg/mL) and penicillin (50 U/mL), shaking at 220 rpm at 22°C. For collecting samples at different developmental times, we harvested the cells at log phase by centrifugation and washed them once with KK2 buffer (16.3 mM KH2PO4, 3.7 mM K2HPO4, pH 6.2). We deposited 5×107 cells onto a 5 cm nitrocellulose membrane placed on a pad saturated with 2 mL PDF buffer (9.2 mM K2HPO4, 13.2 mM KH2PO4, 20 mM KCl, 1.2 mM MgSO4, pH 6.5). We incubated the cells at 22°C in a dark humid chamber untill the specified developmental time.
|
| 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 24 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)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
wildtype at 18-hr, replicate=2
|
| 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 "_expr_raw.tab" contain raw expression values while files ending with "_expr_norm.tab" contain normalized expression values.
|
| |
|
| Submission date |
Mar 27, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Marko Toplak |
| E-mail(s) |
marko.toplak@fri.uni-lj.si
|
| Organization name |
University of Ljubljana
|
| Street address |
Trzaska cesta 25
|
| City |
Ljubljana |
| ZIP/Postal code |
1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL16888 |
| Series (1) |
| GSE45555 |
abc transporters in Dictyostelium discoideum development |
|
| Relations |
| SRA |
SRX256348 |
| BioSample |
SAMN01993546 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1109477_wildtype_r2_18hr_expr_norm.tab.gz |
112.6 Kb |
(ftp)(http) |
TAB |
| GSM1109477_wildtype_r2_18hr_expr_raw.tab.gz |
48.1 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
