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| Status |
Public on Oct 01, 2023 |
| Title |
Roots, protoplasted, rep 2 |
| Sample type |
SRA |
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| Source name |
7 days-after sowing seedling 3 cm roots tips
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| Organism |
Solanum lycopersicum |
| Characteristics |
cultivar: M82
tissue: Whole root tip
genotype: wild type
treatment: Protoplasted
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| Treatment protocol |
In summary, seven days after sowing, 50-100 primary roots per sample were cut ∼3 cm from the root tip. For protoplasted samples, roots were placed in a 35 mm-diameter dish containing a 70 μm cell strainer and 4.5 mL enzyme solution (1.25% [w/v] Cellulase R10, 1.25% Cellulase RS, 0.3% Macerozyme R10, 0.12% Pectolyase, 0.6 M mannitol, 20 mM MES (pH 5.7), 20 mM KCl, 10 mM CaCl2, 0.1% bovine serum albumin, and 0.000194% (v/v) -mercaptoethanol). Cellulase Onozuka R10, Cellulase Onozuka RS, and Macerozyme R10 were obtained from Yakoult Pharmaceutical Industries. Pectolyase was obtained from Sigma-Aldrich (P3026). After digestion at 25°C for 2 hours at 85 rpm on an orbital shaker with occasional stirring, the cell solution was filtered twice through 40 μm cell strainers and centrifuged for 5 minutes at 500 x g in a swinging bucket centrifuge with the acceleration set to minimal. Subsequently, the pellet was resuspended with 1 mL washing solution (0.6 M mannitol, 20 mM MES (pH 5.7), 20 mM KCl, 10 mM CaCl2, 0.1% bovine serum albumin, and 0.000194% (v/v) -mercaptoethanol) and centrifuged for 3 minutes at 500 x g. The pellet was resuspended with 1mL of washing solution and transferred to a 1.7mL microcentrifuge tube.
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| Growth protocol |
Solanum lycopersicum cv. M82 (LA3475) seeds were surface sterilized in 70% (v/v) ethanol for 5 min followed by 50% (v/v) commercial bleach for 20 min and three washes with sterile deionized water. Seeds were plated on 12cmx12cm plates (without sucrose) containing 4.3 g L−1 Murashige and Skoog (MS) medium (Caisson; catalog no. MSP09-50LT), 0.5 g L−1 MES, pH 5.8, and 10 g L−1 agar (Difco; catalog no. 214530) and maintained in a 23°C incubator with 16/8h light/dark periods.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Direct-zol RNA Miniprep kit (ZYMO).
Bulk RNAseq libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Lexogen).
Barcoded libraries were then pooled together, and PE 150-bp reads were sequenced on the NovaSeq 6000 instrument (Illumina) at the UC Davis DNA Technologies Core.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
PROTO2
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| Data processing |
Only read 1 was used in the analyses, since Read2 contained mostly sequencing of the poly A tail of transcripts. Sequences were pooled, and then trimmed and filtered using Trim Galore! (v0.6.6). R1 Trimmed reads were pseudo-aligned to ITAG4.1 transcriptome (cDNA) using Kallisto (v0.46.2), with the parameter -b 100, to obtain count estimates.
Assembly: ITAG4.1 transcriptome (cDNA)
Supplementary files format and content: Column based data frame containing estimated raw pseudo-counts by Kallisto (v0.46.2)
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| Submission date |
Aug 31, 2022 |
| Last update date |
Oct 01, 2023 |
| Contact name |
Siobhan Brady |
| E-mail(s) |
sbrady@ucdavis.edu
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| Organization name |
UC Davis
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| Department |
Plant Biology and Genome Center
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| Street address |
One Shields
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| City |
Davis |
| State/province |
California |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platform ID |
GPL27957 |
| Series (2) |
| GSE212404 |
Protoplast data: suberized exodermis is required for tomato drought tolerance |
| GSE212405 |
Suberized exodermis is required for tomato drought tolerance |
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| Relations |
| BioSample |
SAMN30615193 |
| SRA |
SRX17381387 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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