 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 09, 2021 |
| Title |
DG3913_rep_A [RNA-seq] |
| Sample type |
SRA |
| |
|
| Source name |
whole animal
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype: lin-41(tn1541)
Stage: mid L4
temperature: 20 C
|
| Treatment protocol |
Synchronized populations of developing worms were cultured at 20 °C for 45 hrs after feeding. Harvested worms were washed with water three times and incubated at room temperature for 10 min to allow digestion of intestinal bacteria. Worms were then pelleted by centrifuge at 4,500 rcf for 2 min at room temperature and residual water was removed until the total volumes were twice as the worm pellets. The samples were then flashed frozen by liquid nitrogen and stored at -80 °C.
|
| Growth protocol |
C.elegans were cultured on nematode growth medium (NGM) and fed with E. coli HB101 unless specified. To obtain populations of synchronized developing worms, gravid adults were collected and washed twice with water. Pellets of centrifuged worms were treated with 5 ml 1N NaOH and 1% (v/v) sodium hypochlorite for 5 min with shaking to obtain embryos, and the embryos were rinsed with M9 buffer three times. The embryos were hatched in 10 ml M9 buffer at 20°C for 16-18 hrs with mild shaking. Hatched L1 larvae were transferred to plates at 30-50 worms per plate and replicate plates were cultured for defined periods; samples of the population were examined by microscopy to confirm the developmental stage at the time of harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Harvested worms were washed with M9 medium, centrifuged, and the worm pellets were flash-frozen in liquid nitrogen. The worm pellets were thawed and lysed by adding 4X volumes of QIAzol (Qiagen, Cat: 79306) and shaking vigorously at room temperature for 15 min. The total RNA was extracted by the addition of 0.85X volume chloroform, centrifugation, and recovery of the aqueous phase, which was then re-extracted with 1 volume phenol:chloroform:isoamyl alcohol (25:24:1, pH = 5.5). Total RNA was then precipitated by add 1 volume of isopropanol and 0.5 µl GlycoBlue (Invitrogen, Cat: AM9516), followed by incubation at -80oC for at least 30 min, and recovery by centrifugation at 25,000 rcf for 10 min at 4 °C. The supernatants were then removed, and the RNA pellets were subsequently washed twice by 70% (v/v) ethanol, dried in air for 5 min, dissolved in water, and stored at -80 °C. Worm samples for RNA-seq were aliquoted from the ribosome profiling harvests before the lysis step and frozen separately. The total RNA was extracted as described above. To enrich for mRNA, rRNA was depleted as described in detail elsewhere (Duan et al., 2020b). rRNA-depleted mRNA samples were then purified by RNA Clean & Concentrator-5 Kit (ZYMO, Cat: R1015)
Libraries were constructed by NEBNext Ultra II RNA Library Prep kit (NEB, Cat: E7775, E7335, E7500) and sequenced by Illumina NextSeq 500 system
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
WT for U13A
RNA.raw.reads.DG3913_VT3878.csv
|
| Data processing |
Adaptor removal and size filtering by Cutadapt/1.9
rRNA/tRNA/srpR removal by Bowtie2/2.3.4.3
Genome mapping by Star/2.5.3
Gene counting by featureCounts(subread/1.6.2)
Genome_build: WBCel235
Supplementary_files_format_and_content: comma-seperated values of gene counts
|
| |
|
| Submission date |
Apr 08, 2021 |
| Last update date |
Apr 09, 2021 |
| Contact name |
Ye Duan |
| E-mail(s) |
Ye.Duan@umassmed.edu
|
| Phone |
5088565723
|
| Organization name |
Umass Medical School
|
| Department |
Program of Molecular Medicine
|
| Lab |
Victor Ambros
|
| Street address |
373 Plantation Street, Biotech Two, Suite 306
|
| City |
Worcester |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL19757 |
| Series (2) |
| GSE171733 |
Critical contribution of 3’ non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA [RNA-seq] |
| GSE171748 |
Critical contribution of 3’ non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA |
|
| Relations |
| BioSample |
SAMN18675570 |
| SRA |
SRX10558032 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
