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Status |
Public on Dec 20, 2024 |
Title |
p35S:MIR396, 5DAC, Replicate 3 |
Sample type |
SRA |
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Source name |
Root tips
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Organism |
Arabidopsis thaliana |
Characteristics |
ecotype: Col-0 tissue: Root tips age: 8 days old (cut at 3 days old) genotype: p35S:MIR396 treatment: 5 days after cut
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Treatment protocol |
Control samples were collected ad 3 days old without treatment. For 5DAC samples, 3-day-old seedlings were cut with surgical microblades under a stereoscopic microscope, on agar plates with a nylon mesh of 100µm pore and 70µm fiber. Pores and fibers were used as reference for cut positions. Te samples were collected 5 days after cutting (8 days old).
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Growth protocol |
Sterilized seeds were sown in Petri dishes containing solid medium (Murashige and Skoog salts, 1% sucrose, 0.5g/L MES, 0.8% w/v agar, pH = 5,7) and stratified at 4°C in the dark for at least one day. The plates were placed vertically under continuous light at 22°C.
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Extracted molecule |
total RNA |
Extraction protocol |
Approximately 1000 roots per sample (wild type and p35S:MIR396, uncut controls and 5 days after cut) were collected per quadruplicate for protoplasting. The seeds were sown on nylon mesh, roots were cut at approximately 1cm from the tip, and placed in digesting solution (20mM MES, 400mM mannitol, 20mM KCl, 1.5% cellulase, 0.4% macerozyme, 10mM CaCl2) covered from the light. To enrich the samples with meristematic tissue, the roots were incubated for 20 minutes until the transition zone (between the proliferation and elongation zones) was digested and the tips were released, which was confirmed under microscope. Then, the excess tissue was filtered out using a 1 mm-pore mesh. Digestion continued for 90 minutes and, after filtering the solution twice with a 40µm pore cell strainer, cells were centrifuged down for two minutes at 300rcf and 4°C, washed with washing buffer (enzyme-free digestion solution) and centrifuged again with the same conditions. Buffer was discarded, leaving a small amount at the bottom to resuspend protoplasts, which were then counted in a Neubauer chamber under a light field microscope, and diluted to a concentration of 1000cells/µl. 12000 cells per replicate were sorted on a 10x Chromium platform and libraries were prepared using a 3’ Gene Expression v3.1 kit, following the provider’s instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
NextSeq 550 |
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Description |
JB12 10x Genomics
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Data processing |
Yielded reads were pseudo-aligned to the Arabidopsis genome (TAIR10) using the kallisto bustools software. Seurat matrices were constructed with the Seurat R package. Assembly: TAIR10 Supplementary files format and content: Seurat object
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Submission date |
Feb 21, 2024 |
Last update date |
Dec 20, 2024 |
Contact name |
Julia Lidia Baulies |
E-mail(s) |
baulies@ibr-conicet.gov.ar
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Organization name |
Instituto de Biología Molecular y Celular de Rosario
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Lab |
RNA Biology
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Street address |
Ocampo y Esmeralda s/n
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City |
Rosario |
State/province |
Santa Fe |
ZIP/Postal code |
2000 |
Country |
Argentina |
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Platform ID |
GPL24270 |
Series (1) |
GSE256274 |
microRNA control of stem cell reconstitution and growth in root regeneration |
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Relations |
BioSample |
SAMN40022759 |
SRA |
SRX23687212 |
Supplementary file |
Size |
Download |
File type/resource |
GSM8092422_JB12.seu.RData.gz |
235.0 Mb |
(ftp)(http) |
RDATA |
SRA Run Selector |
Raw data are available in SRA |
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