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Status |
Public on Sep 29, 2022 |
Title |
C. elegans, with bacteria, 0h, rep3 |
Sample type |
SRA |
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Source name |
Head
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Organism |
Caenorhabditis elegans |
Characteristics |
tissue: Head time point: 0h replicate: rep3 genotype: N2 treatment: with bacteria
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Treatment protocol |
For time point 0, dauers were directly collected from the unseeded NGM plate. Dauers assigned to “no bacteria” condition were always collected first and “with bacteria” condition second (10-20 mins later). Assay conditions were the same as for the timelapse imaging experiments and both conditions (no bacteria and + OP50) were processed in parallel. Every hour, a fraction of dauers was washed off the assay plates and anesthetized with 1mM tetramisole. Under a dissecting microscope, the worm’s head was cut off just below the terminal bulb of the pharynx.
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Growth protocol |
Worms were maintained at 15°C on nematode growth medium (NGM) with Escherichia Coli OP50 as food source as previously described. Dauer formation was induced by transferring a mixed population of ca. 50-100 worms to 6cm NGM plates seeded with E. coli OP50. Plates were then incubated for at least 7-10 days at 25°C. Next, worms were washed off with M9, washed twice to eliminate remaining bacteria and 1% SDS solution was added. The mixture was incubated for 30 mins at room temperature (Cassada and Russell, 1975). After an additional washing step with M9 to eliminate SDS, worms were transferred to an unseeded NGM plate. After 30 mins, once the drop of M9 dried out, dauers, which can survive SDS treatment (unlike all other C. elegans stages), crawled out of the mix of dead worms and were collected.
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Extracted molecule |
total RNA |
Extraction protocol |
For each time point 10 heads were pooled into a pre-cooled Eppendorf tube on dry ice, containing 500 µl trizol and 1 µl GlycoBlue. Samples were frozen at -80°C for storage and later subjected to RNA sequencing according to the Tomo-Seq protocol (Holler and Junker, 2019) with minor modifications. Briefly, after thawing, samples were mixed thoroughly and incubated for 5 min at room temperature. Subsequently, 100 µl of chloroform were added to each sample, mixed well and incubated for 5 min. Samples were then centrifuged at 12,000 g for 15 min at 4°C. Next, the aqueous phase was carefully transferred to an Eppendorf LoBind containing 250 µl isopropanol and precipitated overnight at -20°C. Samples were centrifuged for 10 min at 12,000 g and at 4°C. Next, the supernatant was removed and the RNA pellet was washed with 75% freshly prepared ethanol. After washing, the pellet was dried and, once completely dry, resuspended in 2 µl molecular grade water. 1 µl of the sample was transferred to a fresh low binding PCR tube containing barcoded oligo(dT) primer. Reverse transcription was performed with the Superscript II kit according to (Holler and Junker, 2019). First strand synthesis was performed for 1 h at 42°C. Second strand synthesis was performed for 1 h at 16°C. Next, samples were pooled and purified with Agencourt AMPureXP beads. IVT was performed for 16h at 37°C. After fragmentation and purification, RNA was reverse transcribed with superscript II and amplified by PCR. Concentration and size distribution of the resulting cDNA were measured with Qubit and Tapestation. mRNA Libraries were sequenced on a NextSeq500 in paired end mode with 150 cycles.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Raw sequencing basecalls were demultiplexed and converted to FASTQ format using custom python scripts. RNA-seq reads were mapped to the ce11/WBcel235 genome assembly using STAR_2.7.3a (Dobin et al., 2013), annotated using Rsubread (Liao et al., 2019) and count matrices were generated using custom python scripts. Assembly: ce11/WBcel235 Supplementary files format and content: .csv files of read counts per gene
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Submission date |
Sep 26, 2022 |
Last update date |
Oct 01, 2022 |
Contact name |
Friedrich Preusser |
E-mail(s) |
Friedrich.Preusser@t-online.de
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Organization name |
Max Delbrück Center for Molecular Medicine
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Department |
Berlin Institute For Molecular Systems Biology
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Lab |
Preibisch lab
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Street address |
Hannoversche Str. 26
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City |
10115 |
ZIP/Postal code |
Berlin |
Country |
Germany |
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Platform ID |
GPL19757 |
Series (1) |
GSE214208 |
Long-term imaging reveals behavioral plasticity during C. elegans dauer exit. |
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Relations |
BioSample |
SAMN31020234 |
SRA |
SRX17708433 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6601048_FP147_A_0h.csv.gz |
57.6 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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