|
| Status |
Public on Jun 14, 2018 |
| Title |
Replicate2_PIWI-1_high |
| Sample type |
SRA |
| |
|
| Source name |
Sorted PIWI-1 high cells
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
genotype: Wild-type
tissue: Sorted PIWI-1 high cells
treatment: none
|
| Treatment protocol |
1) For RNA-seq of lethal -irradiated animals, GammaCell 40 Exactor irradiator exposed animals to 10,000 rads. 2) For ampuated aniamals, small fragments from one side of planarian were amputated, and whole small regenerating fragments were collected from each time point.
|
| Growth protocol |
Asexual S. mediterranea (strain CIW4) and sexual S2F8 animals were maintained at 20 °C as previously described (Newmark and Sanchez Alvarado, 2000). For all experiments, animals were starved for at least 7 days.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
For untreated and 1-day irradiated animals, total RNA was purified using Trizol as described (Zeng et al., 2013). For sorted X1, X2 and Xins populations based on Hoechst staining, 1x105 cells were sorted into 800 μl Trizol LS, and RNA was extracted according to the manufacturer’s manual. RNA from whole worms was treated with RNase-free DNase on QIAGEN RNAeasy columns and was eluted in nuclease-free water (Ambion). For each replicate, 500ng-1μg RNA from five worms or 100 ng RNA from 1x105 sorted cells were used to generate RNA-Seq libraries using the Illumina TruSeq Stranded kit. For RNA extraction from fixed cells, total RNA was isolated from the pellet using the RecoverAll Total Nucleic Acid Isolation kit (Ambion), starting at the protease digestion stage of manufacturer-recommended protocol. The following modification to the isolation procedure was made: instead of incubating cells in digestion buffer for 15 minutes at 50°C and 15 minutes at 80°C, we carried out the incubation for 1 hours at 50°C. Cell lysates were frozen at −80°C overnight before continuing the RNA isolation by the manufacturer's instructions.
For each replicate, 500ng-1μg RNA per whole worm sample or 100ng of sorted cell RNA was used for generation of RNAseq libraries using the Illumina TruSeq kit. cDNA quality was determined by Agilent high sensitivity DNA kit on Agilent 2100 BioAnalyzer (Agilent Technologies).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
MOLNG-1555
piwi.sorted.fpkm.xlsx
|
| Data processing |
RNAseq reads were aligned to the Schmidtea mediterranea transcriptome smed_20140614 defined in GEO submission GSE72389 using Bowtie2 v2.2.9 default parameters.
Counts were the sum of reads of each transcript. FPKM values were generated using the rpkm function from R package ‘edgeR’.
Genome_build: Schmidtea_mediterranea_3.1
Supplementary_files_format_and_content: FPKM in excel file
|
| |
|
| Submission date |
Dec 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Hua Li |
| E-mail(s) |
hul@stowers.org
|
| Organization name |
Stowers Institute for Medical Research
|
| Street address |
1000 E 50th Street
|
| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL20150 |
| Series (2) |
| GSE107874 |
A tetraspanin family member functionally resolves and facilitates the purification of adult pluripotent stem cells used for whole-body regeneration (Bulk) |
| GSE107875 |
A tetraspanin family member functionally resolves and facilitates the purification of adult pluripotent stem cells used for whole-body regeneration |
|
| Relations |
| BioSample |
SAMN08153302 |
| SRA |
SRX3459787 |
