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| Status |
Public on Jun 26, 2019 |
| Title |
MYB83 Induced Root |
| Sample type |
SRA |
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| Source name |
Root
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: Root
ecotype: Col-0
age: 7-Day Roots
treament: induced with β-estradiol
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| Treatment protocol |
After 7 days of growth plants were transferred by mesh filters to either MS plates buffered to ph 5.8 with KOH or MS containing estradiol (0.01,0.05,0.075,0.1,0.13,0.15,0.175,0.2, 1,2,10, 20uM) plates and grown for an additional 24 hours (induction). All induction experiments were carried out at 7am and collected at 7am. Whole root samples were either imaged, frozen for RNA extraction and qRT-PCR or protoplasted for Drop-seq.
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| Growth protocol |
Seeds were sterilized in a 50% bleach 10% tween solution and then stored at 4°C for 3 days. Sterilized seeds were germinated on nylon mesh (100 uM) filters on square MS petri dish plates and grown at 22° C, 74% humidity in a 12 hr light cycle chamber at a 100-120 light intensity.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Drop-seq runs, plants were grown as described above and roots were harvested then protoplast as previously described (Benfey & Scheres, 2000; K. Birnbaum et al., 2003; Macosko et al., 2015). Individual cells were resuspended in Solution A containing: 600 mM Mannitol, 2mM MgCl2*6H20, 0.10% BSA, 2mM CaCl2*2H20, 2mM MES Hydrate, 10mM KCl and pH to 5.5 with 1M Tris. Each sample of cells was filtered through a 40 μM Cell strainer (BD Falcon) into a 50 mL Falcon tube. Cells were then counted and diluted to 100-180 cells per μl immediately before Drop-seq (Supplemental Table 2).
All Drop-seq steps followed the standard protocol outlined by the McCarroll Lab. The droplet diameter ranged from 0.8 - 0.975 nl (Supplemental Table 2) and Drop-seq was run until 1 mL was collected according to standard Drop-seq protocol. cDNA was amplified to 13-17 cycles and double-purified in 6x volume of Agencourt AMPure XP beads. Tagmentation was carried out with 1200pg of DNA. Samples where ran on the Bioanalyzer (Supplemental Figure 3)
DropSeq + Nextera
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| Library strategy |
RNA-Seq |
| Library source |
genomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
All Drop-seq data was pre-processed and aligned with STAR (Dobin et al., 2013) via the Drop-seq Tools v1.12 software.
Elbow plots where used to determine the best cut-offs for cell numbers
Gene expression matrices were further processed and normalized using the Seurat R package, with a cutoff of at least 500 genes/cell and for genes seen in at least 3 cells
When calculating variable genes cells with a high percentage of mitochondrial, chloroplast and protoplasting induced genes were regressed out variables for downstream clustering
Genome_build: Tair 10
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| Submission date |
May 17, 2018 |
| Last update date |
Jun 27, 2019 |
| Contact name |
Gina Marie Turco |
| E-mail(s) |
gmturco@ucdavis.edu
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| Phone |
5307524656
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| Organization name |
UC Davis
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| Lab |
Brady
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| Street address |
University of California, Davis
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| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platform ID |
GPL19580 |
| Series (1) |
| GSE114615 |
Molecular Mechanisms Driving Switch Behavior in Xylem Cell Differentiation |
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| Relations |
| BioSample |
SAMN09222524 |
| SRA |
SRX4098372 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3145910_star_gene_exon_tagged_800.dge.txt.gz |
3.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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