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Sample GSM6048598 Query DataSets for GSM6048598
Status Public on Jun 10, 2022
Title Mock-infiltrated 20 mins 1000 uE rep 3
Sample type SRA
Source name Mock-infiltrated Arabidopsis leaf 20 mins 1000 uE
Organism Arabidopsis thaliana
Characteristics ecotype: Col-0
condition: High-light (1000 uE)
treatment: Mock
time (min): 20
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.
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.
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description S67_Mock_20_HL_3
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
Submission date Apr 19, 2022
Last update date Dec 18, 2022
Contact name Diep R Ganguly
Phone +1 215-898-0808
Organization name University of Pennsylvania
Department Department of Biology
Lab Brian Gregory
Street address 433 S University Ave
City Philadelphia
State/province PA
ZIP/Postal code 19103
Country USA
Platform ID GPL19580
Series (1)
GSE201015 Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation
BioSample SAMN27628359
SRA SRX14906716

Supplementary file Size Download File type/resource
GSM6048598_67_abundance.h5 2.2 Mb (ftp)(http) H5
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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