|
| Status |
Public on Jun 16, 2011 |
| Title |
EmbryoSingleEnd2 |
| Sample type |
SRA |
| |
|
| Source name |
Ascaris sample
|
| Organism |
Ascaris suum |
| Characteristics |
library preparation: Single end
library id: 2
tissue: mixed samples from 0 - 64 hr embryos
|
| Growth protocol |
For the germinal zone of the testis and ovary, the distal 5 cm of the gonad was isolated from live worms and stored at -80°C. Other regions of the gonads were dissected from live worms and either frozen immediately at -80°C or lysed by homogenization in Trizol and then frozen. Ascaris fertilized eggs were harvested from 4 regions of female uteri (Zygote 1-4) by treatment with 0.5 N NaOH, followed by extensive washing in water, and then stored at 4 °C with 5 volumes of PBS (pH 2.0). For embryonation, the fertilized eggs from final proximal region of the uterus (Zygote-4 = 0 hr embryos) were incubated at 30 °C in PBS (pH 2.0) with constant (100 rpm) shaking for the desired time. The outer shell/membranes were de-coated with 0.4 N KOH/1.4% sodium hypochlorite at 4 °C (zygotes from regions 1-4) or 30°C (developing embryos and larvae) for 180 or 90 minutes, respectively, prior to RNA preparation. RNA from fertilized eggs in the uterus and early embryos (0 hr to 8 days) were extracted directly with Trizol (Invitrogen). Frozen L1, L2, testis and ovary samples were first ground with a mortar and pestle in liquid nitrogen and then extracted with Trizol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
cDNA libraries were prepared from polyadenylated RNA (2x oligo-dT purified using μMACS™ mRNA Isolation Kit, Miltenyi Biotec). Two types of libraries were prepared: 1) sheared, full-length cDNA synthesized using a combination of oligo-dT and random hexamer priming and 2) cDNA prepared from RNA first chemically sheared and then double-stranded cDNA prepared using ramom hexamer priming. For the chemically sheared library, RNA was treated for 75-150 sec with RNA fragmentation buffer (Ambion) at 70°C to generate RNA fragments 150-400 nt in length. First stand cDNA was prepared by random priming (3-6 µg random hexamers/1 µg of A+ RNA) and second stranded cDNA synthesis was carried out using a SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen). Solexa paired-end adaptors were added to the blunt cDNA as described by Illumina, cDNA fragments of 300-400 bp in length were gel purified, the fragments amplified using 17 PCR cycles, and paired-end, 76-120 nt reads were generated as described on the Illumina GAII.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Ascaris mixed samples from 0 - 64 hr embryos
|
| Data processing |
Following removal of the adaptor sequences from the sequence reads, high quality reads with >= 36 nt in length were selected.
|
| |
|
| Submission date |
Jan 29, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jianbin Wang |
| E-mail(s) |
Jianbin.Wang@ucdenver.edu
|
| Phone |
303-724-3227
|
| Organization name |
University of Colorado Denver
|
| Department |
Biochemistry and Molecular Genetics
|
| Lab |
Richard E. Davis Lab
|
| Street address |
12801 East 17th Ave
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL11672 |
| Series (2) |
| GSE26956 |
Ascaris suum transcriptome (RNA-Seq) data |
| GSE26957 |
Deep Small RNA Sequencing from the Nematode Ascaris Reveals Conservation, Functional Diversification, and Novel Developmental Profiles |
|
| Relations |
| SRA |
SRX039743 |
| BioSample |
SAMN00199300 |
