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| Status |
Public on Jun 19, 2022 |
| Title |
Polysome mRNA fraction at 0 mins high-light rep 3 |
| Sample type |
SRA |
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| Source name |
Arabidopsis thaliana 0 mins high-light
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
condition: 0 mins high-light
treatment: Unstressed
time (min): 0
molecule subtype: Polysome-bound mRNA
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| Treatment protocol |
Cordycepin infiltration: 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 |
total RNA |
| Extraction protocol |
mRNA-sequencing: 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).
polysome profiling: Polysome-associated mRNA sequencing was performed using an adapted protocol (Lecampion et al. 2016). Briefly, 250 mg of frozen and ground plant tissue was dissolved in 1 mL of polysome extraction buffer [160 mM Tris-Cl (pH 7.6), 80 mM KCl, 5 mM MgCl2, 5.36 mM EGTA (pH 8), 0.5% IGEPAL CA-630, 40 U/mL RNasin Plus RNase inhibitor (Promega), 150 µg/mL cycloheximide, and 150 µg/mL chloramphenicol] and incubated on ice for 10 minutes. Samples were repeatedly centrifuged at 16,000 rcf for 5 minutes at 4°C until the supernatant was clear. Samples were loaded onto sucrose gradients, consisting of layers of 50% (1.68 mL), 35% (3.32 mL), 20% (3.32 mL) and 20% (1.68 mL), with the two 20% layers added separately (Lecampion et al. 2016). The buffer used in the gradients consisted of 400 mM Tris-Cl (pH 8.4), 200 mM KCl, 100 mM MgCl2, 10.12 µg/mL cycloheximide and 10.12 µg/mL chloramphenicol. Gradients were centrifuged at 41,000 rpm with an SW41Ti rotor for 2 hours at 4°C. Each gradient was passed through a spectrophotometer from highest to lowest density and absorbance at 260 nm recorded, with 1 mL fractions collected. Fractions were pooled into monosomal and polysomal sets and extracted using 5:1 acid phenol:chloroform. Briefy, an equal volume of the phenol:chloroform mix was added to each fraction, before centrifugation at 16,000 rcf for 10 minutes. The aqueous layer was extracted a second time using a one-fifth volume of chloroform, before the RNA was precipitated using an equal volume of isopropanol in addition to sodium acetate (pH 5.2) to a final concentration of 300 mM. The precipitated RNA was washed twice with 70% ethanol and resuspended in a 1 mM sodium citrate (pH 5.4) solution. Total RNA was extracted from finely ground tissue (as above) 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 |
S19_poly_0_3
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| Data processing |
mRNA-sequencing: 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).
Polysome profiling: Quality control of raw reads was carried out using FastQC, with adapter trimming performed using scythe (-p 0.1) and quality trimming performed using sickle (-q 20 -l 20). Reads were aligned to the TAIR10 Arabidopsis genome using subjunc to report a single unambiguous mapping location per read (Liao et al. 2013). Sorting, indexing, and compression was carried out with samtools (Li et al. 2009) and read counts per loci were calculated using featureCounts [-s 2 for reverse stranded libraries, (Liao et al. 2014)]. Reads mapping to rRNA were removed and both normalised read counts of reads per kilobase per million reads (RPKM) and differential gene expression analysis were calculated using edgeR (Robinson et al. 2010). Relative polysomal loading (RPL) was calculated by dividing the RPKM of polysome-bound RNA by that of total RNA on a per replicate basis.
Assembly: TAIR10
Supplementary files format and content: h5: HDF5 file containing run info, abundance esimates (TPM), bootstrap estimates, and transcript length, .counts: gene-level raw counts.
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| Submission date |
Jun 16, 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 |
SAMN29146523 |
| SRA |
SRX15725636 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6249128_Exp566_19_poly.counts.txt.gz |
1.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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