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| Status |
Public on May 17, 2023 |
| Title |
aboveground plant parts, mock-treatment (PBS), biol. rep. 1, exp.1 |
| Sample type |
SRA |
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| Source name |
aboveground plant parts
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: aboveground plant parts
ecotype: Col-0
treatment: mock (PBS)
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| Treatment protocol |
Bacterial suspensions were prepared as previously described in (Miebach et al., 2020). Briefly, bacteria were cultivated at 30 °C on minimal media agar plates containing 0.1% pyruvate as a carbon source. Bacterial suspensions were prepared from bacterial colonies suspended in phosphate-buffered saline (PBS, 0.2 g/L NaCl, 1.44 g/L, Na2HPO4 and 0.24 g/L KH2PO4) and washed twice via centrifugation at 4000 × g for 5 min followed by discarding the supernatant and again adding PBS. The optical density (OD 600 nm) was adjusted so that the suspension contained 2 × 10^7 colony forming units (CFU)/mL. Next, 200 µL of bacterial solution was sprayed per plant tissue culture box using an airbrush spray gun (0.2 mm nozzle diameter, Pro Dual Action 3 #83406). To obtain a homogeneous coverage, the distance between the airbrush spray gun and the plants was increased by stacking a plant tissue culture box, with the bottom cut off, onto the plant tissue culture box containing the plants being spray-inoculated.
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| Growth protocol |
Plants were grown as previously described in (Miebach et al., 2020). Briefly, sterilised seeds were germinated on ½ MS (Murashige and Skoog medium, including vitamins, Duchefa, Haarlem, Netherlands) 1% phytoagar (Duchefa) filled pipette tips. Healthy looking seedlings were aseptically transferred, without removal from the pipette tip, aseptically into Magenta boxes (Magenta vessel GA-7, Magenta LLC, Lockport, IL, USA) filled with ground Zeolite (sourced from cat litter - Vitapet, Purrfit Clay Litter, Masterpet New Zealand, Lower Hutt, New Zealand) and watered with 60 mL ½ MS. Each box received four seedlings. The boxes were closed with lids that allowed for gas exchange and placed into a climate cabinet (85% relative humidity, 11 h light, 13 dark, 21 °C, 150-200 µmol light intensity). Plants were grown for four weeks before they were treated with bacteria or mock controls.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The plant material was collected in RNase-free microcentrifuge tubes (MCT-150-C, Axygen, Corning, USA) and was then immediately flash frozen in liquid N2. Two plants from different growth boxes were pooled per tube to form a biological replicate. Three biological replicates were sampled per treatment and time point. Flash-frozen samples were ground to a fine powder in the collection tube using Teflon pestles (General Lab Supply, Lab Supply, Dunedin, New Zealand). RNA extraction was performed using the Isolate II RNA Plant kit (Bioline, London, England).
RNA library preparation and sequencing was performed by Custom Science (Epping, Australia). RNA quality and purity was assessed using a Nanodrop (ND-2000 Spectrophotometer, Thermo Scientific, Waltham, MA, USA). RNA integrity was verified on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) using the RNA 6000 Nano Kit (Agilent). Poly-A enriched libraries were prepared using the NEBNext UltraTM RNA Library Prep Kit (New England Biolabs, Ipswich, USA) for Illumina. RNA sequencing was performed on a NovaSeq 6000 (Illumina, San Diego, USA) platform. Approximately 30,000,000 paired-end reads with a length of 150 bp were generated per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
MOCK_1
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| Data processing |
Adapters and low-quality reads (end sequences with base quality <20 and sequences with N content > 10%) were removed from raw sequences and sequences below 75 bp were filtered using cutadapt (v1.9.1) (Martin, 2011).
The resulting reads were mapped against the Arabidopsis thaliana reference genome (TAIR10).
Mapped reads were counted with featureCounts (v1.22.2) (Liao et al., 2014).
Assembly: TAIR10
Supplementary files format and content: The file ‘counts_leafBac.txt’ contains the raw counts of sequencing reads for all samples belonging to the different bacterial leaf coloniser experiment.
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| Submission date |
May 11, 2023 |
| Last update date |
May 17, 2023 |
| Contact name |
Moritz Miebach |
| E-mail(s) |
miebach.mor@gmail.com
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| Phone |
+64 2041694314
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| Organization name |
University of Canterbury
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| Department |
School of Biological Sciences
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| Street address |
20 Kirkwood Avenue, University of Canterbury Private Bag 4800
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| City |
Christchurch, Canterbury |
| State/province |
Canterbury |
| ZIP/Postal code |
8041 |
| Country |
New Zealand |
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| Platform ID |
GPL26208 |
| Series (2) |
| GSE232251 |
Non-pathogenic and pathogenic leaf-colonising bacteria elicit qualitatively similar responses |
| GSE232254 |
Non-pathogenic leaf-colonising bacteria elicit pathogen-like responses in a colonisation density-dependent manner |
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| Relations |
| BioSample |
SAMN35036314 |
| SRA |
SRX20287502 |
| 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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