|
| Status |
Public on Mar 12, 2010 |
| Title |
AX4-20-whole organism-1 |
| Sample type |
SRA |
| |
|
| Source name |
AX4 whole organism cell type
|
| Organism |
Dictyostelium discoideum |
| Characteristics |
strain: AX4
supporting culture organism: Klebsiella aerogenes
developmental time point: 20
cell type: whole organism
biological replicate: 1
poly selection: 1X
|
| Growth protocol |
We grew cells to mid-log phase in association with Klebsiella aerogenes bacteria on SM-agar plates. To induce development, we collected the cells, washed them as described, deposited them on nitrocellulose filters and developed them in the dark at 22°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At each time point we collected 1X108 cells directly into 1 ml Trizol reagent (Life Technologies) and extracted total RNA according to the manufacturer’s recommended protocol. To prepare cDNA we subjected 20 µg of total RNA to one round of PolyA selection on oligo(dT) beads (Dynal). We fragmented 125 ng of the resulting mRNA to an average size of 200 bases using divalent cations (Fragmentation Buffer, Ambion) at 70°C for 5 minutes and terminated the reaction with the stop buffer (Ambion). We precipitated the fragments by adding NaOAC, pH 5.2, glycogen and 70% (final concentration) Ethanol, washed the precipitate once with 70% ethanol and resuspended it in RNAse free water. We prepared first-strand cDNA with super script II reverse transcriptase (Invitrogen) and 3µg of random hexamer primers. We then synthesized second strand cDNA with DNA Polymerase I and RNaseH in a illumina (Illumina) custom buffer. We purified the products on a QiaQuick PCR column (Qiagen) and eluted them in 30 μl EB buffer (Qiagen). We further processed the cDNAs using the Genomic DNA Sequencing Sample Prep Kit (Illumina) according to the manufacturer’s recommended protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
dd_AX4_all_20_1.coverage
library selection: total RNA was subjected to one round of PolyA selection on oligo(dT) beads (Dynal)
|
| Data processing |
We sequenced the cDNA libraries to 35 bases on an Illumina high-throughput Illumina Genome Analyzer II using the standard manufacturer’s pipeline (versions 1.2 and 1.3). The resulting FASTQ files were mapped in multiple steps using a short-read alignment software from Novocraft according to the manufacturer’s default parameters (http://www.novocraft.com). First the reads were mapped to reference genome. Sequence reads from D. discoideum were mapped to the May 13, 2009 build of D. discoideum from dictyBase (http://dictybase.org), with the second duplicated region of chromosome 2 (nucleotide 3015984 to 3768555) and the latter half of the RDNA palindrome (nucleotide 42801 to 78150) masked. Sequence reads from D. purpureum were mapped to the D. purpureum genome assembly (manuscript in preparation). Second, all sequences that did not map to the chromosomal sequences were mapped to a library of all possible splice junctions that we have determined using the annotated gene models. The gene models for D. discoideum are defined by the May 13, 2009 build from dictyBase (http://dictybase.org) and D. purpureum by the published genome annotations (manuscript in preparation). The nucleotide coverage was calculated as the sum of all reads starting at a given position.
File naming convention: [species]_[strain]_[cell type]_[time point]_[biological replicate] where dd = D. discoideum and dp = D. purpureum
-- *.coverage files contain coverage for each nucleotide on the forward (F) and reverse (R) strands across the entire genome. Gene models can be found in the supplement of the publication.
|
| |
|
| Submission date |
Aug 13, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Anup Parikh |
| E-mail(s) |
anup.parikh@gmail.com
|
| Phone |
713-798-8308
|
| Organization name |
Baylor College of Medicine
|
| Department |
Molecular & Human Genetics
|
| Lab |
Shaulsky Lab
|
| Street address |
One Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL9379 |
| Series (1) |
| GSE17637 |
Transcriptome conservation of the D. discoideum and D. purpureum developmental programs revealed through RNA-sequencing |
|
| Relations |
| SRA |
SRX015013 |
| BioSample |
SAMN00007120 |
