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| Status |
Public on Jun 10, 2022 |
| Title |
Mock-infiltrated 0 mins 1000 uE rep 2 |
| Sample type |
SRA |
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| Source name |
Mock-infiltrated Arabidopsis leaf 0 mins 1000 uE
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
condition: High-light (1000 uE)
treatment: Mock
time (min): 0
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| Treatment protocol |
The transcriptional inhibitor, cordycepin (3'-deoxyadenosine, Sigma-Aldrich), was syringe-infiltrated on the abaxial side of fully-expanded leaves (true leaves 4-6) of 21-day old plants. Individual leaves were infiltrated with 0.1 mL of incubation buffer [1 mM PIPES (pH 6.25), 1 mM sodium citrate, 1 mM KCl, 15 mM sucrose (Seeley et al. 1992)] with 0.6 mM cordycepin, or without (mock), using a 1 mL needleless syringe (Terumo). Excess liquid was removed from the leaf surface and plants were incubated for a minimum of 10 minutes before further treatment and harvesting. Light-stress was induced by increasing the light intensity to 10× growth irradiance (i.e. 1000 μmol photons m-2 s -1), resulting in a “hot high-light” treatment that effectively induces oxidative stress (Jung et al. 2013). For recovery, plants were returned to pre-stress light conditions.
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| Growth protocol |
Arabidopsis seeds were sown onto moist soil (Seed Raising and Cutting Mix, Martins, NSW, Australia) supplemented with 1 g/L dry volume of soil Osmocote Exact Mini slow release fertilizer (Scotts, NSW, Australia) and 1 L of 0.3 % (v/v) AzaMax (OCP, NSW, Australia). Seeds were covered with a plastic hood and stratified at 4 °C in the dark for at least 72 hours to break dormancy and coordinate germination. Stratified seeds were transferred to a temperature controlled Conviron S10H growth chamber (Conviron, Winnipeg, MB, Canada), fitted with a mixture of 250 W metal halide lamps (Venture Lighting, MH 250W/U) and high pressure sodium lamps (Phillips, SON‐T 250 W E E40 SL/12), for cultivation under a 12-hour photoperiod (08:00-20:00) of 80-100 μ mol photons m-2 s -1, 22.5/20.5 °C day/night temperatures, and 57/55 % day/night relative humidity.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Frozen tissue was ground into a fine powder using a ⅛” steel ball bearing with 1 min shaking at 25 Hz in a TissueLyser II (Qiagen). Total RNA was extracted from finely ground tissue using TRI reagent at a ratio of 1 mL solution per 100 mg ground tissue (Sigma-Aldrich, #T9424-200ML). Residual phenol was removed from the crude extract through two chloroform extractions at a ratio of 1:5 v/v, followed by precipitation using isopropanol at 1:1 v/v. Precipitated RNA was washed twice with 70% ethanol and resuspended in a 1 mM sodium citrate buffer (pH 5.4). RNA quantification was performed through spectrophotometric analysis at 260 nm using the Nanodrop ND-1000 Spectrophotometer and RNA quality was assessed using 1% agarose gel electrophoresis or on the LabChip GX Touch (PerkinElmer).
PolyA-enriched RNA-sequencing libraries were constructed using the Illumina TruSeq Stranded mRNA kit as per the manufacterer's instruction, except reaction volumes were scaled-down by 1/3.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
S65_Mock_0_HL_3
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| Data processing |
Raw read quality was first diagnosed using FastQC (v0.11.7).
Trim Galore! (v0.4.4) was used for adapter and low-quality read trimming with PHRED score < 20 (-q 20).
Kallisto index (-k 21) was used to build the an index from transcripts defined in the cDNA reference annotation (Arabidopsis_thaliana.TAIR10.cdna.all.fa)
Transcript abundance was quantified as TPM using kallisto quant with flags --rf-stranded --bias --single -b 10 -l 300 -s 100 (Bray et al 2016).
Assembly: TAIR10 Ensembl release 51
Supplementary files format and content: h5: HDF5 file containing run info, abundance esimates (TPM), bootstrap estimates, and transcript length
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| Submission date |
Apr 19, 2022 |
| Last update date |
Dec 18, 2022 |
| Contact name |
Diep R Ganguly |
| E-mail(s) |
dganguly@sas.upenn.edu
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| Phone |
+1 215-898-0808
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| Organization name |
University of Pennsylvania
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| Department |
Department of Biology
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| Lab |
Brian Gregory
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| Street address |
433 S University Ave
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19103 |
| Country |
USA |
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| Platform ID |
GPL19580 |
| Series (1) |
| GSE201015 |
Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation |
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| Relations |
| BioSample |
SAMN27628361 |
| SRA |
SRX14906714 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6048596_65_abundance.h5 |
2.2 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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