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| Status |
Public on Sep 14, 2022 |
| Title |
Arabidopsis root cells - sc_44 |
| Sample type |
SRA |
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| Source name |
Arabidopsis root cells - Benfey Lab
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| Organism |
Arabidopsis thaliana |
| Characteristics |
name: WT BRZ then 30 mins BL
genotype: WT
full genotype: WT Col-0
treatment: BL
concentration: 100nM
age: 7_day
timepoint: 0.5
time trt: 0.5_hour_BL
rep: 2
date: 20200212
seq run: Nolan_6199 (NextSeq); Nolan_6226 (NovaSeq S4)
10x chemistry: v3
number of_cells: 6843
number of_genes_detected: 22046
median umi_counts_per_cell: 6935
median number_of_genes_detected_per_cell: 2441
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| Treatment protocol |
For BL scRNA-seq, we first grew plants on 1 μM BRZ to deplete endogenous BRs, then transferred plants to either a fresh BRZ plate or 100nM BL. We monitored the efficacy of these treatments using a constitutively expressed 35S:BES1-GFP line. In agreement with previous reports (Gampala et al., 2007; Ryu et al., 2007; Yin et al., 2002), BES1-GFP was predominantly present in the cytoplasm under low BR conditions resulting from BRZ treatment but accumulated in the nucleus following BL treatment (Figures S1A and S1B). We performed two separate BL scRNA-seq treatment experiments. The first consisted of a BRZ and 2 hour BL treatment. The second experiment included two additional replicates of BRZ and BL 2 hours along with the other time points in our time course (BL 0.5, 1, 4, and 8 hour treatments). Each of the BL treatments was staggered so that all samples were collected simultaneously.WT, bri1-T, and pGL2:BRI1-GFP/bri1-T were similarly profiled in a side-by-side scRNA-seq experiment under control conditions with two replicates per genotype. Lastly, scRNA-seq was performed on WT, gtl1, df1, and gtl1 df1 in duplicate under control conditions.
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| Growth protocol |
Arabidopsis accession Columbia-0 (Col-0) was used as wild type. The following lines were previously described: bri1 GABI_134E10 (Jaillais et al., 2011); bri1-116brl1brl3 triple mutant (bri1-T) (Irani et al., 2012); pGL2:BRI1-GFP/bri1-T (Vragović et al., 2015); gtl1-1 (WiscDsLox413-416C9), df1-1 (SALK_106258), and gtl1-1 df1-1 (Shibata et al., 2018); JKD-Ypet recombineering line (Moreno-Risueno et al., 2015). Seeds were sterilized using 50% (v/v) bleach with 0.05% Tween-20 for 10-15 minutes, plated on 1/2 Linsmaier and Skoog (LSP03-1LT, Caisson Labs; pH 5.7), 1% sucrose media, and stratified 2-4 days at 4°C in the dark. Plates were kept vertically in a Percival growth chamber set to 22°C, 16 hours light/8 hours dark and grown for 7 days. Chemical treatments were conducted by cooling the growth media to approximately 60°C after autoclaving and adding DMSO (a mock solvent), 1μM Brassinazole (BRZ, SML1406, Sigma) or 100nM Brassinolide (BL, 21594, Cayman Chemical).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For each sample, ~0.5cm root tips were harvested from 1000-3000 roots and placed into a 35mm petri dish containing a 70 μm cell strainer and 4.5mL enzyme solution (1.5% [w/v] cellulase [ONOZUKA R-10, GoldBio], 0.1% Pectolyase [Sigma P3026], 0.4 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). The digestion was incubated on an 85 rpm shaker at 25°C for one hour with additional stirring every 15-20 minutes. The resulting cell solution was filtered twice through 40 μm cell strainers and centrifuged for 5 minutes at 500g in a swinging bucket rotor. The pellet was washed with 2mL washing solution (0.4 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), centrifuged again at 500g for 3 minutes, and the pellet resuspended in washing solution at a concentration of ~2000 cells/uL. We loaded 16,000 cells, with the aim to capture 10,000 cells per sample with the 10X Genomics Chromium 3` Gene expression v3 or v3.1 kits.
Cell barcoding and library construction were performed following the manufacturer’s instructions. cDNA and final library quality were verified using a Bioanalyzer High Sensitivity DNA Chip (Agilent) and sequenced on an Illumina NovaSeq 6000 instrument to produce 100bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Processed by COPILOT (https://github.com/ohlerlab/COPILOT)
Assembly: TAIR10
Supplementary files format and content: Seurat object
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| Submission date |
Aug 29, 2022 |
| Last update date |
Sep 17, 2022 |
| Contact name |
Philip N. Benfey |
| Organization name |
Duke University
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| Department |
Biology
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| Lab |
Benfey Lab
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| Street address |
130 Science Drive
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| City |
Durham |
| State/province |
North Carolina |
| ZIP/Postal code |
27708 |
| Country |
USA |
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| Platform ID |
GPL26208 |
| Series (1) |
| GSE212230 |
Single-cell RNA-seq of brassinosteroid response in Arabidopsis roots |
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| Relations |
| BioSample |
SAMN30559708 |
| SRA |
SRX17302366 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6514332_sc_44_COPILOT.rds.gz |
3.2 Gb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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